Nutrition, Metabolism & Cardiovascular Diseases (2014) 24, 328e335

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White fish reduces cardiovascular risk factors in patients with metabolic syndrome: The WISH-CARE study, a multicenter randomized clinical trial C. Vázquez a,r,*, J.I. Botella-Carretero a,r, D. Corella b,r, M. Fiol c,r, M. Lage d,r, E. Lurbe e,r, C. Richart f,r, J.M. Fernández-Real g,r, F. Fuentes h,r, A. Ordóñez i,r, A.I. de Cos j,r, J. Salas-Salvadó k,r, B. Burguera l,r, R. Estruch m,n,r, E. Ros o,p,r, O. Pastor q,r, F.F. Casanueva d,r On behalf of the WISH-CARE Study Investigators a Department of Clinical Nutrition and Obesity, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain b Department of Preventive Medicine, School of Medicine, University of Valencia, Valencia, Spain c Department of Surgical Cardiology, Hospital Universitario Son Espases, Instituto Universitario de Investigación en Ciencias de la Salud de la Universitat de les Illes Balears, Palma de Mallorca, Spain d Division of Endocrinology, Santiago de Compostela University, Santiago de Compostela, Spain e Hospital General de Valencia, Spain f Department of Medicine, Hospital Universitario “Juan XXIII”, Universitat Rovira i Virgili, Tarragona, Spain g Department of Endocrinology, Hospital Josep Trueta, Girona, Spain h Unidad de Lípidos y Arteriosclerosis, IMIBIC/Hospital Universitario Reina Sofía, Universidad de Córdoba, Spain i Department of Endocrinology, Instituto Social de la Marina, Servicio Sanitario de Asistencia Remota de Ibermutuamur, Madrid, Spain j Department of Obesity, Hospital Universitario La Paz, Madrid, Spain k Human Nutrition Unit, Hospital Universitari de Sant Joan de Reus, IISPV, Universitat Rovira i Virgili, Reus, Spain l Division of Endocrinology, Hospital Son Dureta, Universitat Illes Balears, Palma de Mallorca, Spain m Department of Internal Medicine, Hospital Clínic, Barcelona, Spain n Department of Medicine, Universidad de Barcelona, Barcelona, Spain o Unit of Lipids, IDIBAPS Hipertensión, Lípidos y Riesgo Cardiovascular, Hospital Clínic, Barcelona, Spain p Department of Medicine, Universidad de Barcelona, Hospital Clínic, Barcelona, Spain q Department of Biochemistry, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, Spain r CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III (ISCIII), Spain

Received 11 June 2013; received in revised form 12 August 2013; accepted 6 September 2013 Available online 1 November 2013

KEYWORDS Cardiovascular risk; Metabolic syndrome; White fish; Blood pressure; Waist circumference; LDL cholesterol

Abstract Background and aims: Reduction of cardiovascular risk with high consumption of fish in diet is still a matter of debate, and concerns about heavy metal contamination have limited consumption of oily fish. We aimed to evaluate the effect of regular ingestion of white fish on cardiovascular risk factors in patients with metabolic syndrome. Methods and results: Multicenter randomized crossover clinical trial including 273 individuals with metabolic syndrome. An 8-week only-one dietary intervention: 100 g/d of white fish (Namibia hake) with advice on a healthy diet, compared with no fish or seafood with advice on a healthy diet. Outcomes were lipid profile, individual components of the metabolic syndrome, serum insulin concentrations, homeostasis model of insulin resistance, serum C-reactive protein and serum fatty acid levels. We found a significant lowering effect of the intervention with white fish on waist circumference (P < 0.001) and diastolic blood pressure (P Z 0.014). A significant lowering effect was also shown after the dietary intervention with fish on serum LDL concentrations (P Z 0.048), whereas no significant effects were found on serum HDL or triglyceride concentrations. A significant rise (P < 0.001) in serum EPA and DHA fatty acids was observed following white fish consumption. Overall adherence to the intervention was good

* Corresponding author. Department of Clinical Nutrition and Obesity, Hospital Universitario Ramón y Cajal, Ctra. Colmenar, Km 9,100, 28034 Madrid, Spain. Tel.: þ34 913368056; fax: þ34 913368809. E-mail address: [email protected] (C. Vázquez). 0939-4753/$ - see front matter ª 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.numecd.2013.09.018

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and no adverse events were found. Conclusion: In individuals with metabolic syndrome, regular consumption of hake reduces LDL cholesterol concentrations, waist circumference and blood pressure components of the metabolic syndrome. Clinical trial registry: White Fish for Cardiovascular Risk Factors in Patients with Metabolic Syndrome Study, Registered under ClinicalTrials.gov Identifier: NCT01758601. ª 2013 Elsevier B.V. All rights reserved.

Introduction Regular consumption of fish has been reported to decrease coronary heart disease mortality [1], although not in all studies [2]. Current cardiovascular (CV) guidelines for healthy individuals encourage the consumption of fish, preferably oily types, at least twice a week [3] in view of their high content on long-chain n-3 fatty acids (n-3 LCFA). Studies show that regular consumption of n-3 LCFA may reduce the risk of arrhythmia and endothelial dysfunction, and decreased circulating triglyceride levels and inflammation [4]. However, when focusing on the consumption of n-3 LCFA supplements, recent results suggest that there is insufficient evidence of a preventive effect against overall CV events when compared to placebo in primary and secondary prevention [5,6]. These controversial data may be due to the different doses of n-3 LCFA required to produce different effects. The antiplatelet, antiinflammatory, and triglyceride-lowering effects require relatively high doses (3e4 g/day), whereas some of the antiarrhythmic effects, reduction of sudden cardiac death, and improvement in heart failure can be achieved at lower doses (500e1000 mg/day) [7]. Another recent meta-analysis indicate moderate inverse associations between fish consumption and n-3 LCFA and stroke risk, but not so strong with supplements [8]. It is therefore possible that the potential benefit of fish consumption could, in addition to n-3 LCFA, be attributed to a wider array of nutrients (and their interactions) that is abundant in fish. For example, fish is a good source of vitamins D and B complex, which have been linked to inverse CV risk [9,10]. Besides, the positive impact of increasing fish consumption may produce a concomitant reduction in the intake of other foods which may be detrimental to CV health, such as saturated fat; and fish consumption might also be associated with other components of the Mediterranean diet [11]. These data favors the propositions that the future nutritional guidelines should be principally “food based”. They also underscore scientific gaps in the experimental evidence, specifically the lack of studies involving interventions targeting fish intake rather than using supplements, which may have different mechanistic effects. Clinical trials with oily fish have shown beneficial effects on some CV risk factors [12,13], but several concerns have been raised about its contamination with mercury and other heavy metals [14], precluding a wide recommendation for its frequent consumption. Some white fish species, such as hake, although less fatty, have also a high

content of n-3 LCFA and low heavy metals contaminants [15,16] therefore making this type of fish a very interesting one for health. Therefore, the aim of the present study is to test the effect of consuming Namibia hake regularly on lipid profile and the individual components of metabolic syndrome (MetS). Methods Study objectives and participants This study is a multicenter randomized crossover clinical trial conducted on patients with MetS comparing the effect on several CV risk factors of two 8-week dietary interventions: a) advice on a healthy diet avoiding fish or seafood, or b) advice on a healthy diet supplemented with 100 g/d of white fish (Namibia hake). The study was conducted by different centers from Spain belonging to the CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn). MetS was defined by the Third Report of the National Cholesterol Education Program Adult Treatment Panel III criteria [17]. Exclusion criteria were the following: patients taking n-3 LCFA supplements, fish allergy and positive antibodies to Anisakis spp., presence of a body mass index (BMI) 40 kg/m2, chronic kidney disease, liver failure, chronic psychopathy, neoplasia or refusal to participate in the study. Although immigrants were not excluded, in the final population included all subjects were Caucasian. Thirteen centers from ten cities of Spain participated in this study and recruited a total of 273 patients between 2010 and 2011 (see Supplementary data). The protocol was approved by the ethics committee of the Hospital Universitario Ramón y Cajal, and the Institutional Review Boards at all study locations. All participants signed an informed consent. The study was registered at ClinicalTrials.gov Identifier: NCT01758601. Interventions After the screening visit, patients were randomized to one of two sequences: 1) Sequence 1 to receive fish consumption first, and then no fish. The individuals randomized to this arm continued with their previous alimentary habits, avoiding any significant nutritional imbalance as well as any fish or seafood other than the ingestion of 7 serves of hake (each serve consisted of 100 g of frozen Namibia hake, Pescanova S.A., Pontevedra, Spain) per week

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for a period of 8 weeks. Afterward they continued for another 8 weeks with the same diet except for the avoidance of fish and any other seafood (but with no decrease in the total calories or protein ingestion); 2) Sequence 2 for which they started with their previous alimentary habits, avoiding any significant nutritional imbalance, as well as any fish or seafood for the first 8 weeks. Afterward they were changed to the same diet but with 7 serves of hake per week. Patients were maintained with their previous medications throughout the study with no change in doses or type of drug. They also received instructions as not to change their lifestyles, especially regarding exercise. Outcomes For the present study the primary outcome was serum lipids. Secondary outcomes included the other components of the MetS (waist circumference, blood pressure, and fasting blood glucose), LDL cholesterol and C-reactive protein concentrations, and insulin resistance measured by the homeostasis model assessment (HOMA). Serum fatty acids profile was measured as a biomarker of fish consumption. Protocol and data acquisition Patients were evaluated at baseline and after completion of each of the 8-week periods of intervention. Blood samples were obtained after a 12-h overnight fast and were immediately frozen and stored at 25  C until analysis. Lipid profile and fasting glucose were analyzed using an Abbott Aeroset Automated Instrument Analyzer (Abbott Laboratories, Abbot Park, IL). Fasting insulin was measured by immunochemiluminescence (Immulite 2000, Diagnostic Products Corporation, Los Angeles, CA). High sensitive C-Reactive Protein was determined in a c8000 architect autoanalyzer (CRP-VARIO, Abbott Park, IL). BMI, blood pressure (BP) and waist circumference (WC) were also recorded. WC was measured as the smallest perimeter between the costal border and the anterior suprailiac spines, and always by the same investigator at each center. BP was determined in duplicate by manual sphygmomanometer readings in the sitting position and the mean of the two determinations was recorded. At baseline, each patient undertook a semiquantitative food frequency questionnaire (FFQ), which was previously validated for the Spanish population, in order to assess their baseline food consumption habits [18]. A randomized representative sample of patients (n Z 86) was selected in order to measure serum fatty acids (see Supplementary data). As reported by the manufacturer, a mean ingestion of 100 g of hake provides 159.2 mg of eicosapentaenoic acid (EPA) and 482.6 mg of docosahexaenoic acid (DHA) (mean composition from three filets, two medallions and two centers or hake per week) [19]. Therefore, changes in these fatty acids were analyzed in order to assess adherence to the protocol. The fatty acid concentrations, including EPA and DHA, were

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measured as previously reported [20]. Analytical procedures which include C-reactive protein, insulin, lipid profile and fatty acid concentrations were centralized at the laboratory of the coordinating center. Specimens were transported by a specialized and refrigerated service from every center, and stored at 80  C in the Biobank of Hospital Universitario Ramón y Cajal until final laboratory analysis. Sample size, randomization and implementation A priori power analysis was performed using the Ene 3.0 software (http://www.e-biometria.com) with the data obtained from a pilot study with healthy individuals whose lipid levels were compared after taking different amounts of white fish (non-published data). In a crossover study with two groups, at least 88 patients in each group would be required to reach a significant difference of 30 mg/dL in serum triglycerides (TGs) levels with a joint SD of 50 mg/dL and a power of 80%, taking into account the possibility of a maximum 15% of dropouts. Given the nature of the multicenter study and the aim of performing a sub-analysis focusing on the individual components of the MetS and other secondary end-points, we increase the total sample size by more than 30%. After a screening visit and one week of washout to assure an adequate adherence to a balanced diet, participants were randomly assigned to the intervention group using sealed opaque envelopes to yield two groups equally distributed in each participant center. The investigators, who prepared the envelopes and assigned participants to their groups, had no contact with the patients throughout the study. The investigators recruiting the patients, administering the interventions and evaluating the outcomes had no role on the randomization process. Trained dietitians at each center gave personalized dietary advice to participants in order to follow a balanced healthy diet [21] during the study with two periods differing on the consumption of 100 g/d of white fish (Namibia hake) or avoiding every type of fish or seafood. Every patient came to the research center to obtain the frozen hake for the corresponding intervention period. This was given free of charge and provided by the manufacturer directly to the research center. An information brochure of several examples of cooking procedures for the product was also given to each participant during the interview with an expertise dietician. Compliance to the intervention was assured by trained dietitians at each visit, and also with phone contacts during the study. Participants were discontinued if they took more than one serving of any fish or seafood per week in the period of no-fish, or took more than fifteen servings of fish or less than thirteen for two weeks in the other period. Statistical analyses Data are presented as means  SD, unless otherwise stated. Logarithm or square root transformations were applied to ensure normality. For normally distributed

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Outcomes

outcomes, the comparisons between interventions were evaluated with Student t-tests by using the crossover analysis tools of NCSS 2004 software (Number Cruncher Statistical Systems). Baseline characteristics were analyzed by independent t-test, ManneWhitney U test or chi-square test as needed. Statistical analyses were performed by using SPSS for Windows, version 15 (SPSS, Inc., Chicago, Illinois). P values less than 0.05 were considered statistically significant.

There was a significant lowering effect of the intervention with fish (seven servings of fish per week vs. no fish or seafood) on serum LDL concentrations (treatment effect P Z 0.048, period effect P Z 0.23, carryover effect P Z 0.66) (Fig. 2), whereas no significant effects were found on serum HDL or TGs concentrations (Supplementary data) when the analysis was performed per protocol. Similar results were obtained with an intention to treat analysis (Supplementary data). There was a significant lowering effect of the intervention with fish (seven servings of hake per week vs. no fish or seafood) on WC (treatment effect P < 0.001, period effect P Z 0.40, carryover effect P Z 0.81) and diastolic BP (treatment effect P Z 0.014, period effect P Z 0.22, carryover effect P Z 0.05) (Fig. 2). There was a significant period effect for BMI, showing a reduction with time, independently of the intervention (P Z 0.025), and no significant effects were found for the rest of variables in the whole sample of patients (Supplementary data). For the representative sample of patients selected to measure serum fatty acids, there was a significant rise, as expected, in serum EPA (treatment effect P < 0.001, period effect P Z 0.99, carryover effect P Z 0.41), DHA (treatment effect P < 0.001, period effect P Z 0.80, carryover effect P Z 0.43) and omega-3/omega-6 ratio (treatment effect P < 0.001, period effect P Z 0.78, carryover effect P Z 0.19) with the intervention (Fig. 2). Similar results were obtained with an intention to treat analysis (Supplementary data).

Role of the funding source Frozen hake from Namibia was provided free of charge by Pescanova S.A. (Pontevedra, Spain). Funding from Consellería de Economía e Industria 09CSA009E was given to Pescanova S.A. Funding from the CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn) was employed for analytical procedures and transportation of serum specimens. The funding sources had no role in the study design; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the paper for publication. Results A total of 273 patients were initially recruited and randomized, 146 to the sequence of fisheno fish, and the other 127 to the sequence no fishefish. Participant’s baseline characteristics are shown in Table 1. Sixteen individuals were dropouts, so a final sample of 257 patients completed the study, 136 beginning by the fish-no fish sequence, and the rest (n Z 121) by the sequence no fishefish (Fig. 1). Data were analyzed per protocol but also by intention to treat with the last observation carried forward. No differences in the frequency of consumption of white fish, fatty fish, nuts or vegetable oils were observed between sequences of intervention (Table 2).

Adherence and adverse events Sixteen patients were lost to follow-up after the baseline evaluation. Patients were contacted by phone and they communicated their desire not to go on with the protocol, although no adverse event was found in those patients. For

Table 1 Baseline clinical and biochemical characteristics of participants.

Gender; female/male (n) Age (years) Body mass index (kg/m2) Waist circumference (cm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Fasting glucose (mg/dL) Fasting insulin (mU/mL) HOMA insulin resistance C-reactive protein (mg/dL)

Per protocol analysis (n Z 257)

Intention to treat analysis (n Z 273)

Sequence 1 e fisheno fish (n Z 136)

Sequence 2 e no fishefish (n Z 121)

Sequence 1 e fisheno fish (n Z 146)

Sequence 2 e no fishefish (n Z 127)

70/66 56.9  10.6 33.00  4.5 108.7  10.5 140.7  17.4 84.1  9.7 197.5  38.9 46.9  14.1 120.1  42.8 174.2  99.9 128.8  43.9 13.3  13.3 4.38  5.98 4.16  5.30

55/66 58.0  9.6 32.4  4.0 108.1  10.3 140.5  19.9 83.7  11.2 195.19  41.9 44.7  10.9 118.0  34.6 166.7  87.8 126.8  40.5 12.5  9.6 3.62  2.98 6.28  11.25

73/73 56.7  10.8 32.9  4.7 108.3  10.7 140.6  17.4 84.1  9.7 198.7  39.9 47.2  14.1 120.5  42.0 174.6  100.5 128.6  44.8 13.1  13.0 4.26  5.82 4.10  5.17

57/70 57.9  9.4 32.3  4.0 107.9  10.3 140.3  19.9 83.7  11.0 196.4  42.9 45.0  11.2 119.0  35.1 165.9  86.3 126.6  39.9 12.7  9.6 3.68  2.97 6.15  10.99

Data are means  SD, except for gender that is expressed as %. There were no significant differences after independent t test or chi-square test as needed.

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Figure 1 Flow of study. *The individuals on the fish intervention continued with their previous alimentary habits, avoiding any significant nutritional imbalance, and with an ingestion of 7 serves of hake (each serve consisted of 100 g of frozen Namibia hake, Pescanova S.A., Pontevedra, Spain) per week. The no fish intervention consisted of 8 weeks with the same diet except for the avoidance of fish and any other seafood.

the remaining participants, adherence was good and no violation of the pre-established protocol was found (including adherence to diet as checked by expertise dietitians). The concomitant rise in omega-3 fatty acids found with the intervention, and the fall observed when diet with no fish or seafood was followed, talks in favor of the overall good adherence. Table 2 Baseline food frequency consumption. Sequence 1 e fisheno fish (n Z 136) White fish (servings per week) Fatty fish (servings per week) Seafood (servings per week) Nuts (servings per week) Olive oil (servings per week) Other vegetables oilsa (servings per week) Margarine (servings per week) Butter (servings per week)

Sequence 2 e no fishefish (n Z 121)

P value

2.0  1.9

1.8  1.5

0.562

0.6  0.5

0.7  0.6

0.319

0.4  0.6

0.4  0.7

0. 617

5.1  8.6

5.8  9.4

0.937

17.9  10.0

16.9  10.0

0.474

0.9  1.7

1.2  3.3

0.852

0.7  1.7

0.8  2.1

0.562

2.0  1.9

1.8  1.5

0.827

Data are means  SD. a Sunflower seed, corn, or soybean oils.

Discussion We have shown that regular ingestion of white fish in the form of seven servings per week of hake reduces some CV risk factors in patients with the MetS: serum LDL, WC and diastolic BP. These effects were significant, and although not of a big clinical magnitude, taken all together are relevant because only one specific modification to the diet was made. The fact that an expert dietitian monitoring was employed throughout the trial, as to ensure a good adherence to the protocol, gives further evidence that a single dietary modification was responsible for the encountered effects. This adherence was further confirmed by measuring serum fatty acids in a representative subgroup of patients, confirming a rise in EPA and DHA during the periods of fish ingestion. Previous results have shown a decrease in BP levels after intervention with white fish [22]. The beneficial effect of fish on BP has often been ascribed to the increased intake of long-chain omega-3 fatty acids, but fish protein has also been suggested to affect BP levels in animals [23]. Furthermore bioactive peptides possessing hypotensive properties by inhibition of angiotensin converting enzyme have been identified in several fish species [24]. No significant changes in serum triglyceride concentrations were observed in our study, although it was initially expected, as in previous studies fish as a source of EPA and DHA lowered triglyceride concentrations [25].

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Figure 2 Selected outcomes which showed significant differences between interventions. Red dots represent means for the group randomized to sequence 1 (fisheno fish) and blue dots for the group randomized to sequence 2 (no fishefish). Error bars represent standard error of means. All represented variables showed significant differences for treatment effects, and no differences for period or carryover effects (see text for details). EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid.

However, mean intake of EPA and DHA was lower in our study than that previously reported to lower TGs, since 2e4 g/d is the recommended amount for this purpose [26]. Serum LDL reduction in our study may not be a direct effect of fish contents in omega-3 fatty acids, although a previous study has shown some effect on serum LDL concentrations with a high fish diet [27]. We believe that, by a reduction in the intake of saturated fats from other

protein sources, patients were able to lower their LDL concentrations during the period of fish intake. Our results also support that a regular consumption of seven servings of hake per week reduces WC in patients with the MetS. Ours is an intervention study with no bias from other alimentary habits or other confounders. However, as in the case of serum LDL, we cannot rule out the possibility that the beneficial effects found on WC could be due to a

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lowering of saturated fats from meat or other protein sources when on fish intervention. It is noteworthy to remark that the patients in our study lost some weight in the followup but with no differences between groups, so caloric intake did not play a role in the differences found in WC. We found no changes in CRP in our study, as previously reported when fish oil supplementation was given in more physiological levels [28]. We found no effects on serum glucose or insulin concentrations, or even in insulin resistance measured by HOMA, in agreement with a recent meta-analysis [29]. Finally, a limitation of our study is that we did not put the patients into a strict diet, but let them follow a basic balanced alimentary pattern instead. Therefore, the only intervention was the high consumption of hake or no fish at all. This is a good “proof of principle” for a clinical trial, but is more difficult to generalize as the observed results would only benefit to those individuals no eating fish at all who start a high fish consumption pattern. On the other hand, it is noteworthy the fact that the results were observed in a relatively short-term intervention of only 8 weeks, and without introducing any other changes in diet. As regular fish consumption is one of the characteristics of a healthy Mediterranean diet, its effects on CV risk factors may contribute to the overall benefits observed with the Mediterranean diet on CV risk and mortality [30]. In conclusion, regular consumption of hake (seven servings per week) reduces some CV risk factors in patients with the MetS e WC, diastolic BP and LDL levels e and is able to significantly raise the serum omega-3 fatty acid concentrations. Future larger trials are needed to confirm our results. Acknowledgments We want to particularly acknowledge the patients enrolled in this study for their participation and Dr E. Caso and S. Daponte for their initial support. We also thank the Hospital Universitario Ramón y Cajal-IRYCIS Biobank, integrated in the Spanish Hospital Biobanks Network (RetBioH; www.redbiobancos.es) and the Hospital Universitario Son Espases Biobank for their collaboration. No Author declares a conflict of interest. See the supplementary data online for a complete list of rest of Authors/Investigators and Collaborators.

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[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17]

[18]

Appendix A. Supplementary data [19]

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.numecd.2013.09.018. [20]

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