To assess the effect of a 4-week herring diet compared to a reference diet on biomarkers for cardiovascular disease in obese subjects.
Randomized crossover trial.
Department of Internal Medicine, Sahlgrenska University Hospital.
Fifteen healthy obese men and women (age 24–70 years) included, 13 completed.
Subjects were randomly assigned to four weeks of herring diet (150 g baked herring fillets/day 5, days/week) or reference diet (pork and chicken fillets) and switched diets after 2 weeks washout. P-total cholesterol, p-TAG, p-HDL, p-HDL2, p-HDL3, p-LDL, p-apolipoprotein A, p-apolipoprotein B, p-Lipoprotein (a), p-fibrinogen, p-C- reactive protein and p-antioxidative capacity were analysed at 0,2,4,6,8 and 10 weeks.
P-HDL was significantly higher after the herring diet period compared to after the reference diet period; 1.22 vs 1.13 mmol/l (P=0.036). There was a small, but not statistically significant, decrease in TAG but no effect on other biomarkers. TEAC and FRAP, but not ORAC-values, indicated that plasma antioxidants may have been reduced. CRP tended to be lower after the herring diet compared to after the reference diet.
Consumption of oven-baked herring (150g/day, 5 days/week) for 4 weeks, compared to consumption of pork and chicken fillets, significantly increased p-HDL. Patients with insulin resistance and obesity, who commonly have low HDL, may therefore benefit from addition of herring to the diet.
Region Västra Götaland, National board of fisheries (Dr 223-2451-01), Sweden (EU structural funds), The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (Grant No 2001-1246).
Epidemiological studies have shown a lower incidence of cardiovascular diseases (CVD) and cardiovascular mortality with frequent intake of fish. This has been confirmed in intervention studies and several risk factors for CVD have shown to be influenced by fish or fish oil consumption (Undeland et al., 2004). Reduction of fasting plasma triglycerides, raised high-density lipoprotein and reduced thrombosis, inflammation and blood pressure are some of the explanations behind the reduced risk for CVD (Kris-Etherton et al., 2002).
The effects of fish oil on classical risk factors for CVD are well documented. Beneficial effects from fish interventions on risk factors for CVD have been shown in healthy men (Fehily et al., 1983; van Houwelingen et al., 1987; Hänninen and Ågren, 1989; Brown et al., 1990; Brown and Roberts, 1991; Gerhard et al., 1991), in men with a moderately elevated CHD-risk (Mori et al., 1994), in postmenopausal women (Jacques et al., 1992) and in healthy men and women (Wolmarans et al., 1993). Unfortunately, many studies on health effects from fish consumption have neglected to specify important facts like species-related seasonal and geographical variations and the way of pretreating and cooking the consumed fish. In a few studies whole fish has shown more pronounced health effects than fish oil (Cobiac et al., 1991) and lean fish has shown similar beneficial effects as fatty fish (Tidwell et al., 1993; Jacques et al., 1995). This might partly be ascribed to fish consumption being part of a healthier lifestyle or that fish substitutes something less healthy. It has also been proposed that components, other than the fish oil, in fish muscle contribute to the reduction of CVD seen after a diet rich in fish (Kromhout et al., 1985). Apart from fat, fish contains proteins, minerals, vitamins and antioxidants that may affect health (Simopoulos, 1997; Savige, 2001; Elvevoll and Österud, 2003). Whether these potential bioactive components function synergistically with n-3 fatty acids has only been investigated in some studies (Savige, 2001; Elvevoll and Österud, 2003).
Herring (Clupea harengus) is a fatty fish that contains high amounts of long-chain polyunsaturated fatty acids, calcium and vitamin D. In contrast to other fish species, herring is one of the species not threatened by excessive fishing. However, only about 40% of the total catch is used for human consumption and the majority is used to produce feed for other fish species like salmon (Fiskeriverket, 2001, 2002). This makes herring a poorly utilized protein source for humans. The Swedish World Wide Fund for Nature recommends people to eat fresh herring from an environmental point of view (Tuominen and Esmark, 2003; Lundberg, 2004). Hence, there are both environmentally related and nutritionally oriented reasons to investigate if herring could have health beneficial properties.
The objective of this study was to evaluate the effect of a herring diet compared to a reference diet with chicken and pork on risk factors for CVD in obese subjects.
Subjects and methods
Fifteen overweight and obese women and men with a mean age of 50.5 (range 24–70) years volunteered for the study; for baseline characteristics see Table 1. No one smoked or was a high-alcohol consumer (>10 units a week). Subjects were considered eligible if healthy, with no chronic or serious disease and willing to eat herring once a day, 5 days a week, for 4 weeks. They were considered ineligible if pregnant, taking blood lipid lowering or anti-inflammatory drugs. No one normally ate fish more than three times a week, and they did not eat any functional food products or other food with high amounts of n-3 fatty acids.
Two subjects were discontinued from the study, one due to difficulties to attend to the study visits and the other due to regular treatment with the anti-inflammatory drug Ibumetin.
The study was a randomized crossover study with a 2 week wash-out period between the intervention periods. The subjects were provided with 10 frozen meals for every 2 weeks and were instructed to eat one of these meals as lunch or dinner per day, 5 days per week, and preferably on weekdays. From 2 weeks before the study until the end, the subjects were told to follow their normal diet, not to eat any functional foods and not more than two fish meals a week except the herring meals. They were told to maintain their weight during this 12-week-long period and body weight was checked regularly. The study was performed from September to the beginning of December to avoid interference of vacations and public holidays. Fasting blood samples were collected every second week.
The herring was caught on the Swedish West coast in the beginning of September 2003. Fresh skin-free herring fillets were delivered from Paul Mattson AB (Ellös, Sweden) the morning after they had been filleted. The fresh pork fillets were delivered from B Larsson (Swedish meat, Göteborg, Sweden) about 3 days after slaughter and fresh chicken fillets were delivered from Torsåsens Chicken (Falkenberg, Sweden) the morning after they had been slaughtered and filleted. The herring, chicken and pork fillets were kept in 4°C and cooked within 2 days after the delivery. All accompanying food items were delivered from ICA Meny (Partille, Sweden).
The herring, pork and chicken fillets were cooked in the oven at 150°C until the center temperature reached protein denaturation temperature of 55, 63 and 68°C, respectively.
The herring or chicken/pork fillets were served with mashed potato, baked potato wedges, pasta and rice and with different sauces and vegetables. The subjects were served five different dishes, with the average nutritional content shown in Figure 1. All accompanying food items were the same in the two diet periods thus providing a similar amount of carbohydrates. The meals, however, differed in fat content and thus also energy content (Figure 1). The average intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was about 3.4 g/day during the herring diet, while the reference diet provided insignificant amounts (Table 2). The food contained low amounts of salt and little or no spices to avoid extra addition of antioxidants.
All the constituents of the portions were weighed and placed in plastic boxes to optimize an even heating of the food during the final heating in the microwave oven. The dishes contained exactly 150 g of baked herring or 120–150 g of baked chicken or pork fillets (references). The portions were then cooled in 4°C, before put into −40°C. All the food was prepared during 2 weeks before the study started, and then kept at −40°C until handed to the subjects. The subjects were handed 10 meals every second week that they kept in their own freezer (−20°C) until consumption. The subjects were given instructions for thawing and heating the ready meals in the microwave, to avoid overcooking.
Outside the intervention, the subjects consumed their normal diet. Compliance was checked by using 24-h dietary recalls every other week and by analysis of fatty acids in plasma.
Compositional analysis of the food
Herring, pork and chicken (raw and baked) were analysed for total lipids and fatty acid composition. Total lipids were extracted with chloroform and methanol using Bligh and Dyer (1959) modified by Lee (Lee et al., 1996). Two-grams of homogenous and pooled samples were then taken from the herring, chicken and pork minces.
Fatty acid composition was determined in the extracted lipids by converting the fatty acids to methyl esters according to a modification of the method of Lepage and Roy (1986). The extracted lipids were solved in 5 ml toluene, and 1 ml of this extract was transferred to tubes with Teflon-lined caps. The fatty acid C:17 was used as an internal standard. Acetyl chloride (10%) was dissolved in methanol and 1 ml of this solution was added to the lipid samples. Methanolysis was performed in tightly closed tubes in a 70°C water bath for 2 h and shaken every half an hour. After cooling the tubes, 200 μl milliQ-water and 2 ml petroleum ether was added. The tubes were shaken and centrifuged mildly (2500 r.p.m.) for 5 min in room temperature. The upper petroleum ether phase was removed to a new tube. This procedure was repeated twice. It was then shaken, centrifuged and the upper phase was again transfered to a new tube. The solvent was evaporated at 40°C under a stream of nitrogen, and the fatty acid residue was dissolved in 500 μl isooctane. Fatty acid methyl esters (FAME) were analyzed on a Hewlet Packard 5890 capillary gas chromatograph (GC) (Waldbronn, Germany) equipped with a Hewlet Packard auto injector 7673 (Waldbronn, Germany) and detected by a flame ionization detector. A DBwax column (30 m × 0.25 mm) from J&W Scientific (Folsom, USA) was used to separate FAME with hydrogen as carrier gas. The initial temperature was 180°C and was elevated by 5°C/min until 250°C. Injection temperature was 300°C and detection temperature 325°C. Borwin (Le Fontanil, France) evaluation chromatography software was used for evaluation of the detected FAME.
Dry weight was calculated by using 5 g of muscle samples dried in 105°C overnight. Samples were then kept in an exicator until a constant weight was obtained. Protein in the raw and baked herring fillets was analysed by the method of Lowry et al. (1951), and modified by Markwell et al. (1978).
Nutritional values of the meals and of the whole diet (from 24-h recalls) was calculated by a software package (Dietist, Kost och Näringsdata AB, Bromma, Sweden) based on data from the Swedish National Food Administration (Livsmedelsverkets livsmedelsdatabas version 04.1.1).
P-cholesterol and p-triacylglycerol (TAG) levels were determined by fully enzymatic techniques. P-HDL was determined after precipitation of p-apolipoprotein apo B-containing lipoproteins. Precipitation with dextran-sulfate was carried out to separate the p-HDL2 and p-HDL3 subfractions (Gidez and Miller, 1982), and p-low-density lipoprotein cholesterol was calculated as described by Friedewald (Friedewald and Lecy, 1972). P-apolipoprotein B (apo B) and p-apolipoprotein A1 (apo A1) were determined by immunoprecipitation enhanced by polyethylene glycol at 340 nm. All these analyses were performed on a Konelab 20 auto analyzer (Thermo Clinical Labsystems, Espoo, Finland). P-lipoprotein (a) (Lp (a)) was measured by an immunoturbidimetric test (DiaSys Diagnostic Systems Gmbh&Co, Holzheim, Germany). P-C-reactive protein (CRP) was measured by an ultra sensitive method (Orion Diagnostica, Espoo, Finland). For p-fibrinogen, the method of Clauss (1957) and the kit Fibrinogen C from Instrumentation Laboratory (SpA, Milano, Italy) were used.
Serum fatty acid composition was determined as FAME by the same method as was used for the food lipids with some small differences. The fatty acids in 100 μl serum were directly converted to methyl esters without prior fat extraction. Two milliliters of toluene and 2 ml of the methanol solution were added to the serum samples.
Three methods for measuring antioxidative capacity in plasma were used. Trolox Equivalent antioxidant capacity (TEAC) was performed with a commercial kit (NX2332, Randox Laboratories Ltd, Great Britain) following the randox total antioxidant status manual. Ferric reducing ability of plasma (FRAP) was analyzed by the method of Benzie and Strain (1996). Oxygen radical absorbance capacity (ORAC-fluorescein) assay was determined by the Davalos method (Davalos et al., 2004). All three methods measure hydrophilic antioxidants.
All statistical comparisons were made between the dietary endpoints. Results are presented as means±s.e.m. Microsoft Exel version 5.1.2600 SP2 (Microsoft Corporation) was used for the calculations. Paired Student's t-test was used to determine the significance. Regression analyses were performed to examine the relationship between variables.
Lipids, proteins and moisture in herring, pork and chicken fillets
Baked herring had a higher lipid content per wet weight than raw herring and a slightly higher content of proteins (Table 2). About 9% weight loss was observed during baking and about one and half percentage of that weight loss was due to protein loss. The chromatographic analysis showed that EPA and DHA made up about 15.9% of the total fatty acids both before and after baking the herring fillets. These results are in accordance with other studies in herring lipid composition (Regulska-Ilow and Ilow, 2002). The average intake of EPA + DHA/day from the herring diet (150 g baked-herring fillets 5 days a week) was about 3.2 g.
Plasma fatty acids and compliance
The increase in plasma EPA/AA ratio, from the start to the end of the periods of herring consumption, confirmed compliance and was independent of whether the subjects started with the herring or the reference diet (Figure 2). At the start of the study, EPA/AA varied between 0.27% and 0.41% of plasma fatty acids, and the range increased during the herring diet to 0.46–0.99%. There were also significant changes in palmitoleic (16:1), stearic (18:0), oleic (18:1) and dihommo-γ-linolenic acids (20:3) (Table 3).
There were very small differences in plasma fatty acid composition after the reference diet and at baseline. The one exception was the monounsaturated fatty acids, which were slightly increased after the reference diet compared to at baseline.
Influence from the herring intervention on risk factors for CVD
There was a statistically significant increase in p-HDL after the herring intervention, both compared to the reference diet and to the baseline values (Figure 3). Higher P-HDL2 was found after the herring diet when compared to after the reference diet (Table 4). There were no other statistically significant changes in biomarkers, although there were tendencies to reduce p-TAG values (Table 4). Figure 4 shows that p-HDL correlated statistically significantly with the percentage of EPA + DHA in plasma after the herring intervention. A trend toward lower CRP after the herring diet compared to the reference diet was indicated, but not statistically significant.
Influence on plasma antioxidant capacity by increased intake of herring
To test whether the n-3 fatty acid rich herring intervention would affect (or lower) the antioxidant capacities of plasma, three methods were used: trolox equivalent (ORAC, TEAC) and FRAP. Values from TEAC and FRAP, but not ORAC, were statistically and significantly reduced after the herring intervention, compared to the reference diet (Table 5). For TEAC and FRAP there was also a significant negative correlation with the age of the subjects.
In the present study an extensive effort was made to document the preparation of the food itself, something which is often missing in this kind of study. For example, attempts were made to record exactly the postmortem age of the muscle sources and to cook the muscles according to the temperature sensitivity of the proteins from herring, pork and chicken. It has been reported that there are differences between the cardiovascular protecting effect from fish depending on the cooking procedure (Mozaffarian, 2005).
Increased HDL after the herring intervention
The increase in p-HDL owing to the intake of herring suggests important possible health benefits from the herring diet, as low HDL is a well-known risk factor for CVD (Miller and Miller, 1975; Gordon and Castelli, 1977). As HDL is generally lower in overweight and obese individuals, which also was the case in this study (baseline average HDL for men was 0.94 mmol/l and for women 1.27 mmol/l), fish consumption may be even more beneficial for these individuals.
Unexpected lack of influence from herring intake on plasma TAG
Marine n-3 fatty acids have been shown to lower TAG in numerous studies, especially in subjects with hypertriglyceridaemia. A low number of subjects was probably the main reason behind the lack of a statistically significant TAG decrease after the herring intervention in this study.
There are however some other possible explanations. First, when n-3 fatty acids are provided through diet, fish replaces another protein source so that there is a multiplicity of food constituents that are changed simultaneously. Harris (1997) proposed that this is the reason for mixed results regarding the effects of fish on biomarkers for CVDs. Second, the subjects in this study differed from the ones in other fish consumption studies in two ways; they were overweight or obese and half the group were women. A few fish/fish oil interventions on obese subjects have been carried out (Jansen and Lopez-Miranda, 1998; Chan and Watts, 2003). In these, TAG levels were lowered when 24 obese men consumed fish oil capsules (4 g/day) for 6 weeks (Chan and Watts, 2003). About 1-g long-chain n-3 fatty acids/day is commonly set as a limit to get the TAG lowering effect, but whether this amount differs with body weight is not investigated. In our study, it seems like the long-chain n-3 fatty acids in plasma are more elevated when a larger dose/body weight is given. Studies on LCPUFA fatty acids in human blood have suggested important differences between men and women (Hagenfeldt, 1975; Pawlosky et al., 2003). This indicates that the knowledge on effects from fatty fish consumption in premenopausal women is lacking, and there may be differences in the response of these women that have influenced the outcome of this study where seven women were included, out of which three were of fertile age.
Antioxidant capacity in plasma
LCPUFA are easily oxidized and theoretically an increased intake of fatty fish rich in LCPUFA could increase the oxidation in the body. This possible negative effect has previously been investigated, but with varying results (Wolmarans et al., 1993; Anttolainen et al., 1996; Gordoa and Renobales, 2002). Studies have shown that a high fish intake does not influence plasma antioxidants (Anttolainen et al., 1996; Hallgren et al., 2001), but also that hydrophobic antioxidants are reduced after a high intake of fish (Wolmarans et al., 1993).
The results from the present study show that plasma TEAC, FRAP and ORAC values vary widely over time and according to interindividual variance, age and sex. The statistically significant decrease in TEAC and FRAP, but not ORAC values, indicates that the plasma antioxidants may have been influenced by the herring intervention. TEAC, FRAP and ORAC only measure hydrophilic antioxidants. Hydrophobic antioxidants must also be taken into consideration to complete the picture of antioxidant changes in plasma owing to, for example, a high intake of LCPUFA.
The present study does not investigate the effect of exchanging saturated fat or badly balanced fat (high n-6/n-3 ratio) with fish fatty acids, only the difference between a low fat meat diet and a herring diet. The herring meals contained more fat and thus more energy than the reference meals, but 24 h recalls indicated that this was partly compensated with a higher intake of other food (carbohydrate-rich). A slight weight gain was observed during the herring intervention compared to the reference period (Table 4). These facts may have influenced the outcome on p-TAG.
The large variation in P-EPA/AA ratio after the herring intervention, compared to baseline and reference intervention (Figure 2), was probably partly due to that the subjects ate 10 intervention meals freely distributed over 14 days. Thus the variation in P-EPA/AA ratio could reflect a small difference in the last day's intake of herring. However, the proportion of EPA and DHA in the plasma is also influenced by the intake of other essential fatty acids as well as of the total amount of fat in the diet. This could explain the wide diversity in changes of EPA, DHA and AA between different individuals (Bezard et al., 1994).
Figure 2 shows that both groups had a higher plasma EPA/AA ratio at the start of the second intervention period (week 6) compared to baseline. This indicates that the 2 weeks of washout was fairly short and that group 2 (that started with the reference diet) tended to eat more food that contains EPA and DHA during the wash-out period in between the two diets than they did before the study start and during the reference diet.
To conclude, intake of oven-baked herring fillets compared to chicken and pork fillets increased p-HDL in plasma of overweight and obese subjects. P-TAG was slightly, but not significantly decreased after the herring diet compared to the reference diet. These results need to be confirmed in a larger and more homogenous group of subjects. Antioxidant capacity, measured by TEAC, FRAP and ORAC in plasma, gave an ambiguous answer and it is therefore not possible to draw any clear conclusions about changes in antioxidative status.
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The herring fillets were supported by Paul Mattsson AB, Ellös, Sweden. Thanks to Annette Almgren for help with both the food preparation and blood sampling and to Nils-Gunnar Carlsson for the methods for fatty acid analysis. Special thanks to Angela Silveira at Karolinska Hospital (Stockholm), who made it possible for us to analyze fibrinogen in the plasma samples.
Contributors and Guarantors: H Lindqvist, AS Sandberg, AM Langkilde and I Undeland.
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Lindqvist, H., Langkilde, A., Undeland, I. et al. Herring (Clupea harengus) supplemented diet influences risk factors for CVD in overweight subjects. Eur J Clin Nutr 61, 1106–1113 (2007). https://doi.org/10.1038/sj.ejcn.1602630
- n-3 fatty acids
- plasma lipids
- high-density lipoproteins
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