M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized crossover trial Keiko Kondo a , Katsutaro Morino a,⁎, Yoshihiko Nishio b , Motoyuki Kondo a , Keiko Nakao a , Fumiyuki Nakagawa a, c , Atsushi Ishikado a, d , Osamu Sekine a , Takeshi Yoshizaki a , Atsunori Kashiwagi a , Satoshi Ugi a , Hiroshi Maegawa a a
Department of Medicine, Division of Endocrinology and Metabolism, Shiga University of Medical Science, Shiga, Japan Department of Diabetes and Endocrine Medicine, Division of Human and Environmental Sciences, Kagoshima University, Graduate School of Medical and Dental Sciences, Kagoshima, Japan c Osaka Laboratory, JCL Bioassay Corporation, Osaka, Japan d R&D Department, Sunstar Inc, Osaka, Japan b
A R T I C LE I N FO Article history:
AB S T R A C T Objective. The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs)
Received 22 October 2013
consumption on atherosclerosis have been reported in numerous epidemiological studies.
Accepted 8 April 2014
However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in
Keywords: Diet
postmenopausal women with type 2 diabetes mellitus (T2DM). Materials/Methods. Twenty-three postmenopausal women with T2DM were assigned to
n-3 PUFA
two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in
Intervention studies
a randomized crossover design. Endothelial function was measured with reactive
Endothelial function
hyperemia using strain-gauge plethysmography and compared with the serum levels of
Diabetes mellitus
fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR). Results. A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fishbased diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/ dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase.
Abbreviations: ADMA, asymmetric dimethylarginine; ANOVA, analyses of variance; DHA, docosahexaenoic acid; DHET, dihydroxyeicosatrienoic acid; EET, epoxyeicosatrienoic acid; eNOS, endothelial nitric oxide synthase; EPA, eicosapentaenoic acid; FBF, forearm blood flow; FDR, flow debt repayment; GPR, G-protein coupled receptor; 4-HHE, 4-hydroxy hexenal; 4-HNE, 4-hydroxy nonenal; HOMA, homeostatic model assessment; hs-CRP, high-sensitivity C-reactive protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MCP-1, monocyte chemotactic protein-1; NOS, nitric oxide synthase; 8-OHdG, 8-hydroxydeoxyguanosine; PUFA, polyunsaturated fatty acid; RH, reactive hyperemia; SBP, systolic blood pressure; sEH, soluble epoxide hydrolase. ⁎ Corresponding author at: Department of Medicine, Division of Endocrinology and Metabolism, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga 520-2192, Japan. Tel.: + 81 77 548 2222; fax: +81 77 543 3858. E-mail address: [email protected] (K. Morino). http://dx.doi.org/10.1016/j.metabol.2014.04.005 0026-0495/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Kondo K, et al, A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized c..., Metabolism (2014), http://dx.doi.org/10.1016/j.metabol.2014.04.005
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ME TA BOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Conclusions. A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
The beneficial effects of dietary fish intake on coronary heart disease, sudden cardiac death, and all-cause mortality in the general population have been discussed for many decades [1]. A negative correlation between the dietary intake of fish and cardiovascular risk has been noted [2–4]. In diabetic women, a higher consumption of fish has been associated with a lower incidence of coronary heart disease and total mortality [5]. Although the mechanisms underlying the beneficial effects of dietary fish intake remain unclear, n-3 polyunsatulated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), might be important factors. Endothelial dysfunction is a major contributor to the pathogenesis of cardiovascular disease. Many studies have indicated that endothelial dysfunction is caused by smoking, obesity, dyslipidemia, hypertension, and type 2 diabetes mellitus [6–9]. Supplementation of n-3 PUFAs has been shown to improve endothelial function in patients with hypercholesterolemia [10], peripheral arterial disease [11], acute myocardial infarction [12], and type 2 diabetes mellitus [13,14]. Few reports have sought to evaluate if a fish-based diet intervention, instead of n-3 PUFA supplements, can improve endothelial function directly. Eicosanoids are generated from other n-3 or n-6 PUFAs and control multiple functions, including inflammation, thrombosis and endothelial function [15]. Increasing evidence has revealed that epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent vasodilatation [16]. Soluble epoxide hydrolase (sEH), which converts EETs to dihydroxyeicosatrienoic acid (DHET), is a potential pharmacological target of endothelial dysfunction [17]. It has been reported that plasma EETs levels and the EET/DHET ratio decreased in patients with hypertension [18]. In addition, one recent study showed that fish oil supplementation increased the levels of some eicosanoids in young, healthy volunteers [19]. However, the mechanism by which a fish-based dietary intervention changes the circulating eicosanoids in subjects with type 2 diabetes mellitus is not known. This study had two aims. First, we examined the effects of a fish-based dietary intervention on endothelial function in postmenopausal women with type 2 diabetes mellitus. Second, we sought to analyze the relationships among endothelial function, n-3 PUFAs, and the concentrations of their metabolites.
3.1 kg/m2) at Shiga University of Medical Science Hospital. Individuals taking pioglitazone, EPA, or fish oil supplements; those undergoing insulin treatment; and those with poor glycemic control (HbA1c ≧ 8.4% [68.3 mmol/mol]), high fish intake (frequency of fish intake ≧ seven times per week), fish allergy, smoking, or excessive alcohol ingestion were excluded. The nature and potential risks of the study were explained to all participants, and written informed consent was obtained. The study was performed in accordance with the principles of the Declaration of Helsinki. The protocol was approved by the ethics committee of Shiga University of Medical Science. The study was registered at UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm) with the Identification No. UMIN000002277.
2.2.
Study design
2.
Materials and Methods
The study included two experimental periods in a randomized crossover design. Participants were randomly assigned to start with either the fish-based diet or the control diet for 4 weeks. Eleven participants (fish-first group) started with the fish-based diet, and 12 participants (control diet-first group) started with the control diet for 4 weeks. After the first experimental period, the participants crossed over to the other experimental diet for an additional 4 weeks. Participants were instructed to take more than 3.0 g/day n-3 PUFA derived from fish (e.g., Pacific saury, salmon, sardines, etc.) during the fish-based diet period. Conversely, during the control diet period, participants were instructed to avoid fish intake, particularly n-3 PUFA-rich fish. The intake of total energy was 126–135 kJ per kg ideal body weight throughout the study period. Participants were advised by a dietician to modify their habitual diet qualitatively before each diet period. At baseline and at the end of each experimental period, a dietician provided participants with written and verbal instructions regarding the completion of a 3-day dietary record with a digital photograph of each meal. Participants also kept a food diary to record their daily fish intake. Nutritional intake, including intake of n-3 PUFA, was calculated using food composition tables specific for the Japanese people (Eiyoukun, ver5.0, Kenpakusha, Tokyo, Japan) [20]. Participants were advised on maintaining a constant physical activity level throughout the study. They underwent a day of testing at baseline and after 4 weeks of each diet intervention. Tests included anthropometric measurements, blood samples, and body composition measurements after a 10–16 h overnight fast. No medications were allowed to be changed throughout the study period.
2.1.
Study population
2.3.
Twenty-three postmenopausal type 2 diabetic women were recruited (mean age, 69.7 ± 6.6 years; mean BMI, 22.5 ±
Weight and body composition
Body weight was recorded with an electronic scale without shoes and with light clothing weighing a maximum of 0.1 kg.
Please cite this article as: Kondo K, et al, A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized c..., Metabolism (2014), http://dx.doi.org/10.1016/j.metabol.2014.04.005
M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
The percentage of body fat was determined by bioelectrical impedance analysis (Bio Electrical Impedance Analyzer System, Tanita, Tokyo, Japan).
2.4. Assessment of endothelial function by strain-gauge plethysmography The endothelial function of each participant was examined after an overnight fast. Each participant was kept in a supine position during this measurement. After 15 min in the supine position, the basal forearm blood flow (FBF) was measured using a mercury-filled silastic (Dow Corning, Midland, MI, USA) strain-gauge plethysmography (EC-5R, D.E. Hokanson, Issaquah, WA, USA), as described by Linder et al. [21]. The effect of reactive hyperemia (RH) on FBF was measured as described in other studies, with minor modifications [22]. To induce RH, FBF was occluded by inflating the cuff on the right upper arm to a pressure of 190 mmHg when the systolic blood pressure (SBP) was 140 mmHg and under, or 50 mmHg plus SBP when the SBP was over 140 mmHg, for 5 min. FBF was measured after release of the cuff until it returned to the basal level. The peak FBF response [21], duration of RH, and total reactive hyperemic flow (flow debt repayment [FDR]) [23] during RH were used to assess the endothelial function of resistance vessels. The percent peak FBF, as an index of the peak FBF response, was obtained by calculating the increment of the peak FBF divided by the mean basal FBF.
2.5.
Laboratory analyses
All qualifying participants underwent a complete medical history evaluation and a physical examination. Blood samples were taken after an overnight fast. Plasma glucose concentrations were measured using the hexokinase glucose 6-phosphate dehydrogenase ultraviolet method. Serum insulin concentrations were measured using a chemiluminescent enzyme immunoassay. Serum triglyceride concentrations were determined enzymatically. Serum total cholesterol concentrations were measured by the cholesterol dehydrogenase ultraviolet method. Serum HDL-cholesterol concentrations were determined by a direct method (Cholestest N HDL, Sekisui Medical, Tokyo, Japan). LDL-cholesterol concentrations were calculated using the Friedewald equation (total cholesterol − [HDL-cholesterol + triglyceride/5]). Insulin resistance was evaluated using homeostatic model assessment (HOMA) and calculated as the product of the fasting glucose and insulin concentrations. HbA1c levels were measured by high-performance liquid chromatography. Serum adiponectin (Otsuka Pharmaceutical, Tokyo, Japan), monocyte chemotactic protein-1 (MCP-1) (R&D Systems, Minneapolis, MN, USA), and asymmetric dimethylarginine (ADMA) (Immundiagnostik, Bensheim, Germany) concentrations were measured by ELISA. Serum high-sensitivity C-reactive protein (hs-CRP) levels were measured by nephelometry. Urine 8-Hydroxydeoxyguanosine (8-OHdG) concentrations were measured by enzyme immunoassays.
2.6.
Measurement of PUFA and metabolites
Serum fatty acid profiles were determined by gas chromatography–mass spectrometry. Serum 4-hydroxy hexenal (4-HHE)
3
and 4-hydroxy nonenal (4-HNE) levels were measured by liquid chromatography–mass spectrometry, as previously described [24] (1 out of 23 samples was removed because of an extremely high value). Eicosanoids in human serum samples were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a modified procedure [25]. After blood sampling by vein puncture, the serum was separated by centrifugation at 1750 g for 10 min at room temperature, and 1000 μL of sample was transferred into polypropylene tubes and stored at –80 °C until measurement. Serum samples as an internal solution (50 ng/mL; PGE2-d4, 6-keto PGF1a-d4 20-HETE-d6 (Cayman Chemical, Ann Arbor, MI, USA) diluted with methanol) were spiked to all samples, including calibration standards. For the preparation of calibration standards, isotonic sodium chloride solution (Otsuka Pharmaceutical Factory, Tokushima, Japan) was used as the surrogate matrix. Into this surrogate matrix, a standard solution of 84 compounds of eicosanoid (Cayman Chemical), diluted with methanol in each concentration (0.05–100 ng/mL), was spiked. Calibration curve samples were extracted using the same procedure as for the serum samples. For the extraction procedure, 2 mL of water was added to all samples for dilution. After vortex mixing, the samples were added to solid-phase extraction cartridges (Empore C18-SD, 3 M, Maplewood, MN, USA). Ethyl acetate was used for elution, and this elute was evaporated under stream nitrogen gas at 36 °C. After being reconstituted with 40 μL of methanol, this aliquot was injected into an optimized liquid chromatography–tandem mass spectrometry system. Liquid chromatography was performed using an ACQUITY UPLC (Waters, Milford, MA, USA), and an API4000 triple quadrupole tandem mass spectrometer (AB Sciex, Foster City, CA, USA) was used as a detector. An analytical column (ACQUITY UPLC HSS T3, 1.8 μm, 2.1 mm × 150 mm, Waters) was used. Column temperature was maintained at 60 °C. Injected samples were eluted using water/acetonitrile/acetic acid (7:3:0.1, v/v/v) and acetonitrile/2-propanol (1:1, v/v), with a linear gradient at a total flow of 0.215 mL/min. To operate the API4000, electronic spray ionization in negative mode with selected reaction monitoring was used. The selected reaction monitoring transition parameter, the database of Lipid Map (http://www.lipidmaps.org/), was referred to for selected precursors to produce ions of m/z, respectively. Other parameters were adjusted to optimum values.
2.7.
Statistical analyses
We previously observed that fish-based dietary intervention increased the peak FBF by 47.0% in 10 healthy volunteers (unpublished data). In this study, we calculated a sample size using these data. As a result, 22 participants would have been significant to detect changes with a power of 80% and a 95% confidence level. Statistical analyses were performed with SPSS version 17.0 (SPSS, Tokyo, Japan, 2008). The distribution of variables was analyzed by checking histograms and normal plots of the data, and the normality was tested using the Kolmogorov– Smirnov and Shapiro–Wilk tests. Changes in nutritional intake were compared for all participants, and metabolic variables, serum fatty acid and endothelial function were compared in each group. For normally distributed data,
Please cite this article as: Kondo K, et al, A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized c..., Metabolism (2014), http://dx.doi.org/10.1016/j.metabol.2014.04.005
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ME TA BOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Table 1 – Physical and clinical characteristics of the subjects at baseline. Variable
All subjects (n = 23)
Fish-first group (n = 11)
Control diet-first group (n = 12)
Age (years) BMI (kg/m2) Body fat (%) SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HbA1c (%) HbA1c (mmol/mol) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Triglyceride (mmol/l) Fish consumption (times/week) Use of glucose-lowering agents, n (%) Use of statin, n (%) Use of anti-hypertensive agents, n (%)
69.7 ± 6.6 22.5 ± 3.1 32.0 ± 5.3 131.3 ± 12.3 73.3 ± 8.2 6.88 ± 1.10 31.6 ± 15.0 6.8 ± 0.4 51.0 ± 4.7 5.71 ± 0.95 1.65 ± 0.31 3.53 ± 0.88 2.65 ± 1.39 5.6 ± 2.5 13 (56.5) 8 (34.8) 9 (39.1)
69.5 ± 6.0 22.0 ± 2.7 30.6 ± 4.3 133.9 ± 6.3 73.2 ± 5.2 7.06 ± 0.94 30.3 ± 14.3 6.9 ± 0.4 51.6 ± 4.5 5.16 ± 0.57 1.52 ± 0.28 3.09 ± 0.52 2.78 ± 1.64 5.1 ± 2.2 6 (54.5) 6 (54.5) 6 (54.5)
69.8 ± 7.3 23.0 ± 3.5 33.4 ± 6.0 129.0 ± 16.0 73.5 ± 10.5 6.71 ± 1.25 32.9 ± 16.1 6.8 ± 0.5 50.5 ± 5.0 6.21 ± 0.97 1.77 ± 0.30 3.93 ± 0.96 2.54 ± 1.18 6.1 ± 2.7 7 (58.3) 2 (16.7) 3 (25.0)
p value 0.919 a 0.447 a 0.209 a 0.341 a 0.938 a 0.457 a 0.684 a 0.563 a 0.563 a 0.019 a 0.053 a 0.037 a 0.686 a 0.330 a 1.000 b 0.089 b 0.214 b
Values are means ± SD for continuous variables. DBP, diastolic blood pressure; SBP, systolic blood pressure. a Unpaired t test. b Chi-square test.
repeated measures analysis of variance (RM-ANOVA) was performed for comparisons of different periods of determinations. In addition, post-hoc testing was performed using Tukey’s honest significance difference test. For variables with non-normal distributions, the non-parametric Friedman test with Wilcoxon test was used as a post-hoc test with Bonferroni correction. Analysis of covariance (ANCOVA) was performed to compare the effects of each dietary treatment. All data are expressed as the mean ± SD for continuous variables. P-values of < 0.05 were considered statistically significant, unless specifically described.
3.
Results
3.1.
Participants and diet diaries
The baseline characteristics of the study participants are shown in Table 1. The percentages of participants using oral glucose-lowering, lipid-lowering, or anti-hypertensive agents were 56.5%, 43.5%, and 39.1%, respectively. No participant stopped or started these medications during the study. There were no differences in potential confounding factors, such as age, BMI and medication, between the 2 groups, although the total and LDL-cholesterol levels were higher in the control diet-first group compared with fish-first group. Nutritional intake was estimated by analyzing diet diaries at baseline, during the fish-based diet period, and during the control diet period (Table 2). The intake of total energy was unchanged throughout the study period but decreased during the control diet period. The intake of protein significantly decreased, and that of carbohydrate significantly increased, during the control diet period compared with the baseline and fish-based diet periods. The intake of fat was unchanged throughout the study period. The intake of fish and n-3 PUFA showed a significant increase during the fish-based diet
period and a decrease during the control diet period, compared with that during the baseline period.
3.2.
Changes in metabolic variables in each group
The changes in metabolic variables during the study period are shown in Table 3 and were analyzed separately for each group. In both groups, the anthropometric and body composition values of the study participants did not change significantly during the study period. In the fish-first group, serum triglyceride concentrations decreased at week 4 of the fish-based diet period and increased at week 8 of the control diet period. Similarly, in the control diet-first group, serum triglyceride concentrations increased at week 4 of the control diet period and decreased at week 8 of the fish-based diet period. HDL-cholesterol concentrations tended to increase after the fish-based diet period and to decrease after the control diet period in both groups. Total cholesterol and LDLcholesterol concentrations did not change significantly during the study period. Fasting plasma glucose, serum insulin, HbA1c levels and HOMA-R were unchanged during the study period. In the control diet-first group, serum adiponectin levels decreased significantly after both the control and fishbased diet periods compared with the baseline period, but not in the fish-first group. Serum MCP-1, hs-CRP, ADMA and urine 8-OHdG levels were unchanged during the study period in both groups (Table 3).
3.3. Change in metabolic variables before and after each diet in all patients The changes in metabolic variables in all patients are shown in Table 4. The anthropometric and body composition values of the study subjects did not change significantly during the study period. The fish-based diet significantly decreased serum total cholesterol (p = 0.013) and triglyceride concentration (p = 0.008)
Please cite this article as: Kondo K, et al, A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized c..., Metabolism (2014), http://dx.doi.org/10.1016/j.metabol.2014.04.005
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M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Table 2 – Nutritional intake at baseline and changes during the intervention. Variable Energy (kJ) Energy (kJ/kg) Protein (% energy) Carbohydrate (% energy) Fat (% energy) n-3 PUFA (g/day) n-3 PUFA (g/day) (from fish) n-6 PUFA (g/day) n-6/n-3 Fish intake (g/day)
Baseline 7508 143 16.9 55.6 25.7 3.2 1.9 9.3 3.6 123.5
± ± ± ± ± ± ± ± ± ±
1116 25 1.9 6.4 5.6 2.0 1.8 3.3 1.9 59.5
Fish-based diet period 7349 139 17.6 54.0 26.6 4.3 3.2 8.5 2.2 159.7
± ± ± ± ± ± ± ± ± ±
1046 22 1.6 5.8 5.5 1.4 c 1.1 2.4 1.0 31.9
Control diet period 7035 134 15.4 58.2 24.7 1.5 0.2 9.4 6.4 44.6
± ± ± ± ± ± ± ± ± ±
p value a
1060 23 1.2 c, f 5.3 e 5.0 0.5 d, f 0.3 2.4 1.2 30.8 d, f
p value b
0.186 0.164
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized crossover trial Keiko Kondo a , Katsutaro Morino a,⁎, Yoshihiko Nishio b , Motoyuki Kondo a , Keiko Nakao a , Fumiyuki Nakagawa a, c , Atsushi Ishikado a, d , Osamu Sekine a , Takeshi Yoshizaki a , Atsunori Kashiwagi a , Satoshi Ugi a , Hiroshi Maegawa a a
Department of Medicine, Division of Endocrinology and Metabolism, Shiga University of Medical Science, Shiga, Japan Department of Diabetes and Endocrine Medicine, Division of Human and Environmental Sciences, Kagoshima University, Graduate School of Medical and Dental Sciences, Kagoshima, Japan c Osaka Laboratory, JCL Bioassay Corporation, Osaka, Japan d R&D Department, Sunstar Inc, Osaka, Japan b
A R T I C LE I N FO Article history:
AB S T R A C T Objective. The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs)
Received 22 October 2013
consumption on atherosclerosis have been reported in numerous epidemiological studies.
Accepted 8 April 2014
However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in
Keywords: Diet
postmenopausal women with type 2 diabetes mellitus (T2DM). Materials/Methods. Twenty-three postmenopausal women with T2DM were assigned to
n-3 PUFA
two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in
Intervention studies
a randomized crossover design. Endothelial function was measured with reactive
Endothelial function
hyperemia using strain-gauge plethysmography and compared with the serum levels of
Diabetes mellitus
fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR). Results. A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fishbased diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/ dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase.
Abbreviations: ADMA, asymmetric dimethylarginine; ANOVA, analyses of variance; DHA, docosahexaenoic acid; DHET, dihydroxyeicosatrienoic acid; EET, epoxyeicosatrienoic acid; eNOS, endothelial nitric oxide synthase; EPA, eicosapentaenoic acid; FBF, forearm blood flow; FDR, flow debt repayment; GPR, G-protein coupled receptor; 4-HHE, 4-hydroxy hexenal; 4-HNE, 4-hydroxy nonenal; HOMA, homeostatic model assessment; hs-CRP, high-sensitivity C-reactive protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MCP-1, monocyte chemotactic protein-1; NOS, nitric oxide synthase; 8-OHdG, 8-hydroxydeoxyguanosine; PUFA, polyunsaturated fatty acid; RH, reactive hyperemia; SBP, systolic blood pressure; sEH, soluble epoxide hydrolase. ⁎ Corresponding author at: Department of Medicine, Division of Endocrinology and Metabolism, Shiga University of Medical Science, Tsukinowa, Seta, Otsu, Shiga 520-2192, Japan. Tel.: + 81 77 548 2222; fax: +81 77 543 3858. E-mail address: [email protected] (K. Morino). http://dx.doi.org/10.1016/j.metabol.2014.04.005 0026-0495/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Kondo K, et al, A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: A randomized c..., Metabolism (2014), http://dx.doi.org/10.1016/j.metabol.2014.04.005
2
ME TA BOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Conclusions. A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
The beneficial effects of dietary fish intake on coronary heart disease, sudden cardiac death, and all-cause mortality in the general population have been discussed for many decades [1]. A negative correlation between the dietary intake of fish and cardiovascular risk has been noted [2–4]. In diabetic women, a higher consumption of fish has been associated with a lower incidence of coronary heart disease and total mortality [5]. Although the mechanisms underlying the beneficial effects of dietary fish intake remain unclear, n-3 polyunsatulated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), might be important factors. Endothelial dysfunction is a major contributor to the pathogenesis of cardiovascular disease. Many studies have indicated that endothelial dysfunction is caused by smoking, obesity, dyslipidemia, hypertension, and type 2 diabetes mellitus [6–9]. Supplementation of n-3 PUFAs has been shown to improve endothelial function in patients with hypercholesterolemia [10], peripheral arterial disease [11], acute myocardial infarction [12], and type 2 diabetes mellitus [13,14]. Few reports have sought to evaluate if a fish-based diet intervention, instead of n-3 PUFA supplements, can improve endothelial function directly. Eicosanoids are generated from other n-3 or n-6 PUFAs and control multiple functions, including inflammation, thrombosis and endothelial function [15]. Increasing evidence has revealed that epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent vasodilatation [16]. Soluble epoxide hydrolase (sEH), which converts EETs to dihydroxyeicosatrienoic acid (DHET), is a potential pharmacological target of endothelial dysfunction [17]. It has been reported that plasma EETs levels and the EET/DHET ratio decreased in patients with hypertension [18]. In addition, one recent study showed that fish oil supplementation increased the levels of some eicosanoids in young, healthy volunteers [19]. However, the mechanism by which a fish-based dietary intervention changes the circulating eicosanoids in subjects with type 2 diabetes mellitus is not known. This study had two aims. First, we examined the effects of a fish-based dietary intervention on endothelial function in postmenopausal women with type 2 diabetes mellitus. Second, we sought to analyze the relationships among endothelial function, n-3 PUFAs, and the concentrations of their metabolites.
3.1 kg/m2) at Shiga University of Medical Science Hospital. Individuals taking pioglitazone, EPA, or fish oil supplements; those undergoing insulin treatment; and those with poor glycemic control (HbA1c ≧ 8.4% [68.3 mmol/mol]), high fish intake (frequency of fish intake ≧ seven times per week), fish allergy, smoking, or excessive alcohol ingestion were excluded. The nature and potential risks of the study were explained to all participants, and written informed consent was obtained. The study was performed in accordance with the principles of the Declaration of Helsinki. The protocol was approved by the ethics committee of Shiga University of Medical Science. The study was registered at UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm) with the Identification No. UMIN000002277.
2.2.
Study design
2.
Materials and Methods
The study included two experimental periods in a randomized crossover design. Participants were randomly assigned to start with either the fish-based diet or the control diet for 4 weeks. Eleven participants (fish-first group) started with the fish-based diet, and 12 participants (control diet-first group) started with the control diet for 4 weeks. After the first experimental period, the participants crossed over to the other experimental diet for an additional 4 weeks. Participants were instructed to take more than 3.0 g/day n-3 PUFA derived from fish (e.g., Pacific saury, salmon, sardines, etc.) during the fish-based diet period. Conversely, during the control diet period, participants were instructed to avoid fish intake, particularly n-3 PUFA-rich fish. The intake of total energy was 126–135 kJ per kg ideal body weight throughout the study period. Participants were advised by a dietician to modify their habitual diet qualitatively before each diet period. At baseline and at the end of each experimental period, a dietician provided participants with written and verbal instructions regarding the completion of a 3-day dietary record with a digital photograph of each meal. Participants also kept a food diary to record their daily fish intake. Nutritional intake, including intake of n-3 PUFA, was calculated using food composition tables specific for the Japanese people (Eiyoukun, ver5.0, Kenpakusha, Tokyo, Japan) [20]. Participants were advised on maintaining a constant physical activity level throughout the study. They underwent a day of testing at baseline and after 4 weeks of each diet intervention. Tests included anthropometric measurements, blood samples, and body composition measurements after a 10–16 h overnight fast. No medications were allowed to be changed throughout the study period.
2.1.
Study population
2.3.
Twenty-three postmenopausal type 2 diabetic women were recruited (mean age, 69.7 ± 6.6 years; mean BMI, 22.5 ±
Weight and body composition
Body weight was recorded with an electronic scale without shoes and with light clothing weighing a maximum of 0.1 kg.
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M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
The percentage of body fat was determined by bioelectrical impedance analysis (Bio Electrical Impedance Analyzer System, Tanita, Tokyo, Japan).
2.4. Assessment of endothelial function by strain-gauge plethysmography The endothelial function of each participant was examined after an overnight fast. Each participant was kept in a supine position during this measurement. After 15 min in the supine position, the basal forearm blood flow (FBF) was measured using a mercury-filled silastic (Dow Corning, Midland, MI, USA) strain-gauge plethysmography (EC-5R, D.E. Hokanson, Issaquah, WA, USA), as described by Linder et al. [21]. The effect of reactive hyperemia (RH) on FBF was measured as described in other studies, with minor modifications [22]. To induce RH, FBF was occluded by inflating the cuff on the right upper arm to a pressure of 190 mmHg when the systolic blood pressure (SBP) was 140 mmHg and under, or 50 mmHg plus SBP when the SBP was over 140 mmHg, for 5 min. FBF was measured after release of the cuff until it returned to the basal level. The peak FBF response [21], duration of RH, and total reactive hyperemic flow (flow debt repayment [FDR]) [23] during RH were used to assess the endothelial function of resistance vessels. The percent peak FBF, as an index of the peak FBF response, was obtained by calculating the increment of the peak FBF divided by the mean basal FBF.
2.5.
Laboratory analyses
All qualifying participants underwent a complete medical history evaluation and a physical examination. Blood samples were taken after an overnight fast. Plasma glucose concentrations were measured using the hexokinase glucose 6-phosphate dehydrogenase ultraviolet method. Serum insulin concentrations were measured using a chemiluminescent enzyme immunoassay. Serum triglyceride concentrations were determined enzymatically. Serum total cholesterol concentrations were measured by the cholesterol dehydrogenase ultraviolet method. Serum HDL-cholesterol concentrations were determined by a direct method (Cholestest N HDL, Sekisui Medical, Tokyo, Japan). LDL-cholesterol concentrations were calculated using the Friedewald equation (total cholesterol − [HDL-cholesterol + triglyceride/5]). Insulin resistance was evaluated using homeostatic model assessment (HOMA) and calculated as the product of the fasting glucose and insulin concentrations. HbA1c levels were measured by high-performance liquid chromatography. Serum adiponectin (Otsuka Pharmaceutical, Tokyo, Japan), monocyte chemotactic protein-1 (MCP-1) (R&D Systems, Minneapolis, MN, USA), and asymmetric dimethylarginine (ADMA) (Immundiagnostik, Bensheim, Germany) concentrations were measured by ELISA. Serum high-sensitivity C-reactive protein (hs-CRP) levels were measured by nephelometry. Urine 8-Hydroxydeoxyguanosine (8-OHdG) concentrations were measured by enzyme immunoassays.
2.6.
Measurement of PUFA and metabolites
Serum fatty acid profiles were determined by gas chromatography–mass spectrometry. Serum 4-hydroxy hexenal (4-HHE)
3
and 4-hydroxy nonenal (4-HNE) levels were measured by liquid chromatography–mass spectrometry, as previously described [24] (1 out of 23 samples was removed because of an extremely high value). Eicosanoids in human serum samples were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a modified procedure [25]. After blood sampling by vein puncture, the serum was separated by centrifugation at 1750 g for 10 min at room temperature, and 1000 μL of sample was transferred into polypropylene tubes and stored at –80 °C until measurement. Serum samples as an internal solution (50 ng/mL; PGE2-d4, 6-keto PGF1a-d4 20-HETE-d6 (Cayman Chemical, Ann Arbor, MI, USA) diluted with methanol) were spiked to all samples, including calibration standards. For the preparation of calibration standards, isotonic sodium chloride solution (Otsuka Pharmaceutical Factory, Tokushima, Japan) was used as the surrogate matrix. Into this surrogate matrix, a standard solution of 84 compounds of eicosanoid (Cayman Chemical), diluted with methanol in each concentration (0.05–100 ng/mL), was spiked. Calibration curve samples were extracted using the same procedure as for the serum samples. For the extraction procedure, 2 mL of water was added to all samples for dilution. After vortex mixing, the samples were added to solid-phase extraction cartridges (Empore C18-SD, 3 M, Maplewood, MN, USA). Ethyl acetate was used for elution, and this elute was evaporated under stream nitrogen gas at 36 °C. After being reconstituted with 40 μL of methanol, this aliquot was injected into an optimized liquid chromatography–tandem mass spectrometry system. Liquid chromatography was performed using an ACQUITY UPLC (Waters, Milford, MA, USA), and an API4000 triple quadrupole tandem mass spectrometer (AB Sciex, Foster City, CA, USA) was used as a detector. An analytical column (ACQUITY UPLC HSS T3, 1.8 μm, 2.1 mm × 150 mm, Waters) was used. Column temperature was maintained at 60 °C. Injected samples were eluted using water/acetonitrile/acetic acid (7:3:0.1, v/v/v) and acetonitrile/2-propanol (1:1, v/v), with a linear gradient at a total flow of 0.215 mL/min. To operate the API4000, electronic spray ionization in negative mode with selected reaction monitoring was used. The selected reaction monitoring transition parameter, the database of Lipid Map (http://www.lipidmaps.org/), was referred to for selected precursors to produce ions of m/z, respectively. Other parameters were adjusted to optimum values.
2.7.
Statistical analyses
We previously observed that fish-based dietary intervention increased the peak FBF by 47.0% in 10 healthy volunteers (unpublished data). In this study, we calculated a sample size using these data. As a result, 22 participants would have been significant to detect changes with a power of 80% and a 95% confidence level. Statistical analyses were performed with SPSS version 17.0 (SPSS, Tokyo, Japan, 2008). The distribution of variables was analyzed by checking histograms and normal plots of the data, and the normality was tested using the Kolmogorov– Smirnov and Shapiro–Wilk tests. Changes in nutritional intake were compared for all participants, and metabolic variables, serum fatty acid and endothelial function were compared in each group. For normally distributed data,
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ME TA BOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Table 1 – Physical and clinical characteristics of the subjects at baseline. Variable
All subjects (n = 23)
Fish-first group (n = 11)
Control diet-first group (n = 12)
Age (years) BMI (kg/m2) Body fat (%) SBP (mmHg) DBP (mmHg) Glucose (mmol/l) Insulin (pmol/l) HbA1c (%) HbA1c (mmol/mol) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) LDL-cholesterol (mmol/l) Triglyceride (mmol/l) Fish consumption (times/week) Use of glucose-lowering agents, n (%) Use of statin, n (%) Use of anti-hypertensive agents, n (%)
69.7 ± 6.6 22.5 ± 3.1 32.0 ± 5.3 131.3 ± 12.3 73.3 ± 8.2 6.88 ± 1.10 31.6 ± 15.0 6.8 ± 0.4 51.0 ± 4.7 5.71 ± 0.95 1.65 ± 0.31 3.53 ± 0.88 2.65 ± 1.39 5.6 ± 2.5 13 (56.5) 8 (34.8) 9 (39.1)
69.5 ± 6.0 22.0 ± 2.7 30.6 ± 4.3 133.9 ± 6.3 73.2 ± 5.2 7.06 ± 0.94 30.3 ± 14.3 6.9 ± 0.4 51.6 ± 4.5 5.16 ± 0.57 1.52 ± 0.28 3.09 ± 0.52 2.78 ± 1.64 5.1 ± 2.2 6 (54.5) 6 (54.5) 6 (54.5)
69.8 ± 7.3 23.0 ± 3.5 33.4 ± 6.0 129.0 ± 16.0 73.5 ± 10.5 6.71 ± 1.25 32.9 ± 16.1 6.8 ± 0.5 50.5 ± 5.0 6.21 ± 0.97 1.77 ± 0.30 3.93 ± 0.96 2.54 ± 1.18 6.1 ± 2.7 7 (58.3) 2 (16.7) 3 (25.0)
p value 0.919 a 0.447 a 0.209 a 0.341 a 0.938 a 0.457 a 0.684 a 0.563 a 0.563 a 0.019 a 0.053 a 0.037 a 0.686 a 0.330 a 1.000 b 0.089 b 0.214 b
Values are means ± SD for continuous variables. DBP, diastolic blood pressure; SBP, systolic blood pressure. a Unpaired t test. b Chi-square test.
repeated measures analysis of variance (RM-ANOVA) was performed for comparisons of different periods of determinations. In addition, post-hoc testing was performed using Tukey’s honest significance difference test. For variables with non-normal distributions, the non-parametric Friedman test with Wilcoxon test was used as a post-hoc test with Bonferroni correction. Analysis of covariance (ANCOVA) was performed to compare the effects of each dietary treatment. All data are expressed as the mean ± SD for continuous variables. P-values of < 0.05 were considered statistically significant, unless specifically described.
3.
Results
3.1.
Participants and diet diaries
The baseline characteristics of the study participants are shown in Table 1. The percentages of participants using oral glucose-lowering, lipid-lowering, or anti-hypertensive agents were 56.5%, 43.5%, and 39.1%, respectively. No participant stopped or started these medications during the study. There were no differences in potential confounding factors, such as age, BMI and medication, between the 2 groups, although the total and LDL-cholesterol levels were higher in the control diet-first group compared with fish-first group. Nutritional intake was estimated by analyzing diet diaries at baseline, during the fish-based diet period, and during the control diet period (Table 2). The intake of total energy was unchanged throughout the study period but decreased during the control diet period. The intake of protein significantly decreased, and that of carbohydrate significantly increased, during the control diet period compared with the baseline and fish-based diet periods. The intake of fat was unchanged throughout the study period. The intake of fish and n-3 PUFA showed a significant increase during the fish-based diet
period and a decrease during the control diet period, compared with that during the baseline period.
3.2.
Changes in metabolic variables in each group
The changes in metabolic variables during the study period are shown in Table 3 and were analyzed separately for each group. In both groups, the anthropometric and body composition values of the study participants did not change significantly during the study period. In the fish-first group, serum triglyceride concentrations decreased at week 4 of the fish-based diet period and increased at week 8 of the control diet period. Similarly, in the control diet-first group, serum triglyceride concentrations increased at week 4 of the control diet period and decreased at week 8 of the fish-based diet period. HDL-cholesterol concentrations tended to increase after the fish-based diet period and to decrease after the control diet period in both groups. Total cholesterol and LDLcholesterol concentrations did not change significantly during the study period. Fasting plasma glucose, serum insulin, HbA1c levels and HOMA-R were unchanged during the study period. In the control diet-first group, serum adiponectin levels decreased significantly after both the control and fishbased diet periods compared with the baseline period, but not in the fish-first group. Serum MCP-1, hs-CRP, ADMA and urine 8-OHdG levels were unchanged during the study period in both groups (Table 3).
3.3. Change in metabolic variables before and after each diet in all patients The changes in metabolic variables in all patients are shown in Table 4. The anthropometric and body composition values of the study subjects did not change significantly during the study period. The fish-based diet significantly decreased serum total cholesterol (p = 0.013) and triglyceride concentration (p = 0.008)
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M ET ABOL I SM CL IN I CA L A N D E XP E RI ME N TAL XX ( 2 0 14 ) XXX– X XX
Table 2 – Nutritional intake at baseline and changes during the intervention. Variable Energy (kJ) Energy (kJ/kg) Protein (% energy) Carbohydrate (% energy) Fat (% energy) n-3 PUFA (g/day) n-3 PUFA (g/day) (from fish) n-6 PUFA (g/day) n-6/n-3 Fish intake (g/day)
Baseline 7508 143 16.9 55.6 25.7 3.2 1.9 9.3 3.6 123.5
± ± ± ± ± ± ± ± ± ±
1116 25 1.9 6.4 5.6 2.0 1.8 3.3 1.9 59.5
Fish-based diet period 7349 139 17.6 54.0 26.6 4.3 3.2 8.5 2.2 159.7
± ± ± ± ± ± ± ± ± ±
1046 22 1.6 5.8 5.5 1.4 c 1.1 2.4 1.0 31.9
Control diet period 7035 134 15.4 58.2 24.7 1.5 0.2 9.4 6.4 44.6
± ± ± ± ± ± ± ± ± ±
p value a
1060 23 1.2 c, f 5.3 e 5.0 0.5 d, f 0.3 2.4 1.2 30.8 d, f
p value b
0.186 0.164
