Foods, nutrients or whole diets: effects of targeting fish and LCn3PUFA consumption in a 12mo weight loss trial

A 12 month randomised controlled trial was conducted between 2009–2010 with overweight adults in three parallel diet advice arms: low calorie + placebo (control); low calorie + fish + placebo (Fish); and low calorie + fish + eicosapentaenoic acid (EPA)/docosahexanoic acid (DHA) supplements (Fish + S). The second intervention arm served as an enhancement strategy in case of poor compliance to fatty fish consumption over the year. The primary outcomes were change in body weight, BMI and percentage body fat. Secondary outcomes included change in cardiovascular disease risk factors. Recruitment was by media advertisements and emails sent by the research team throughout Wollongong, a major coastal city 70 km south of Sydney, Australia. This study was conducted according to Declaration of Helsinki guidelines and procedures were approved by the University of Wollongong Human Research Ethics Committee. Written informed consent was obtained from all participants. This trial is registered at http://​www.​anzctr.​org.​au (ID: ACTRN12608000425392).

A researcher independent of the subject interface undertook the randomisation of participants into diet groups (stratified by sex and block randomised; nQuery Advisor V 7.0, Statistical solutions, Cork, Ireland) and the code was kept from the researchers collecting dietary data and delivering treatment. Supplements were coded off-site. Different dietitians collected dietary data and provided dietary advice. Those providing advice were necessarily aware of diet category but not of supplement allocation.

The study sample comprised middle aged (mean ± SD age 45 ± 10 y) obese adults (mean ± SD BMI 31.3 ± 3.5 kg/m²). Inclusion criteria included: age between 18 to 60 years, BMI between 25 to 37 kg/m², waist circumference > 94 cm (men) or > 80 cm (women), otherwise healthy. Individuals with major illnesses, diabetes mellitus, LDL ≥ 6 mmol/L, food allergies or habits inhibiting compliance, low literacy, inadequate conversational English, and those who are already taking fish oil supplements, unable to undertake study requirements, pregnant/lactating, not weight stable (within 3 kg) in the past 6 months or on a weight-loss diet were excluded from participation. Participants were not paid.

The intervention addressed dietary intake at the, food (fish), key nutrient (LCn3PUFA) and whole diet (low calorie) level. The fatty fish target and LCn3PUFA equivalent reference value were based on materials in the Nutrient Reference Values (NRVs) report for Australia and New Zealand [17]. These suggested two servings (180 g) of fatty fish per week and a dietary intake for LCn3 PUFA based on the 90^th percentile of population consumption levels (i.e. 610 mg/day for men and 430 mg/day for women) [17]. All groups were advised on a hypocaloric diet (2 MJ energy below estimated energy requirements estimated by the Mifflin equation [18] with 1.25 physical activity factor), targeting 25% energy from protein, 45% energy from carbohydrate and 30% energy from fat. Diets referred to low fat staple foods (fruit, vegetables, cereals, lean meat, low fat milk and yoghurt) and small amounts of nuts, seeds, spreads and oils. Food groups were congruent with the Australian Guide to Healthy Eating [19], and formed the basis of the standardised procedure for both diet groups and the template of the diets sheets. Participants in the Fish and Fish + S advice groups were specifically encouraged to consume 180 g fatty fish per week. Participants in the control group were not given specific advice regarding fish intake, but were instructed to consume prescribed amounts of foods rich in protein, of which fish was an option. Dietary education was supported by print resources outlining methods of cooking the appropriate fish and recipes. The Control and Fish groups were given placebo capsules (1 g olive oil per day) and the Fish + S group the active supplements (420 mg EPA + 210 mg DHA; Blackmore’s Promega Heart, Blackmores Australia). Dietary education (1 hour) and follow-up (30 min) with one of seven experienced dietitians was provided at 0, 1, 2, 3, 6, 9, and 12 months with written materials and monthly newsletters to support compliance. Accredited Practising Dietitians collected dietary data at 0, 3 and 12 months via a validated diet history interview (DH) [20]. Dietary data was analysed using the FoodWorks software system (Version 6, 2009, Xyris Software, Spring Hill, QLD, Australia). Reported food intake data was converted to intakes of energy and macronutrients using the AUSNUT1999 [21] and AusFood2001 databases (revision 11, from FoodWorks 2009 version 6, Xyris Software, Spring Hill, QLD, Australia). Reported LCn3PUFA intakes were assessed separately using the AUSNUT2007 database [22]. The percentage fish in each dish or product was calculated based on a validated approximation of canned fish labels [23] and standardized recipe data. Habitual physical activity at baseline and 12 months was assessed by questionnaire [24]. Advice was given to all groups to walk for 30 minutes three days per week.

Height was measured using a stadiometer without shoes. Body weight was measured in an upright position in minimal clothing, without shoes using digital scales with a bioelectrical impedance component (Tanita TBF-622; W.W. Wedderburn Pty Ltd, Ingleburn, NSW, Australia) at baseline and every 3 months until 12 months. Percentage body fat was measured using DEXA (Hologic QDR 4500; Hologic Inc. Bedford MA) from most participants at baseline (n = 104), 3 months (n = 66) and 12 months (n = 46).

Fasting blood samples taken at 0, 3, 6, 9 and 12 months were sent to a quality assured laboratory (Southern IML Pathology) to measure total cholesterol, HDL, LDL, triglycerides, insulin, and glucose. Leptin was assessed by the same laboratory for samples collected at 0, 3 and 12 months. Erythrocyte fatty acid concentrations at 0, 3 and 12 months, a measure of dietary compliance [25], were analysed at Australian Research Laboratories (now Healthscope Pathology), Melbourne, Australia. The omega-3 index was calculated as erythrocyte [EPA + DHA]/[total fatty acids] × 100% [26]. Insulin resistance was assessed by HOMA equations [27]. Blood pressure was measured using a medical grade blood pressure monitor (Dinamap XL Vital Signs Monitor). Participants sat for 10 mins then three readings were taken from the same arm, and the mean of the three readings was used as the final reading.

Compliance to fish and supplement recommendations was calculated for all participants who remained in the trial at 12 months. In order to calculate compliance to fish recommendations, an average of each participant’s fish intake from all available diet histories was taken, as described by Alhassan et al[28]. Compliers were defined as consuming < 180 g fish/week for the control group, consuming ≥ 180 g fish/week for the Fish group, and either consuming ≥ 180 g fish/week or taking ≥ 90% of the supplements in the Fish + S group.

Using data from a previous study [29] and an expected weight change of −2.6 kg in the control and −4.7 kg in the intervention groups, we required 27 participants in each group to achieve statistical significance (α = 0.05, β = 0.9). As shown in Figure 1, we enrolled 126 people, from an initial pool of 374 volunteers. Eight participants withdrew from the study before treatment so their data were excluded from all analyses.

All statistical analyses were performed using IBM SPSS (version 17.0 and 19.0, IBM Australia, Lane Cove, NSW, Australia). Analysis of the primary outcome (i.e. weight) was performed on an intention to treat basis with the 118 participants who provided baseline data, using a linear mixed model. Several approaches were taken to investigate the effect of missing data on the primary outcome. These included multiple imputation (using PROC MI in SAS V9.2, SAS Inc. Cary, NC), last observation carried forward (LOCF), and baseline observation carried forward (BOCF). These methods are the most commonly used in weight loss studies [30]. Completers only (n = 63) analyses were also performed.

In the intention to treat analysis (Table 2) based on the linear mixed model, all groups lost weight at 12 months (Control −4.5 kg vs. Fish −4.3 kg vs. Fish + S −3.3 kg; p < 0.001) and percentage body fat significantly decreased (Control: -1.5% vs. Fish: -1.4% vs. Fish + S: -0.7%; p < 0.001). There were no significant differences between groups for these variables and this result remained after adjustment for EPA + DHA status (Figure 3). All other methods of analysis for the primary outcome similarly showed a significant time effect, with no significant group differences, and restricting the analysis to completers only did not significantly change the results. After stratifying the analysis based on compliance, there was no significant weight loss among non-compliers regardless of dietary assignment (time effect = 0.653; group effect = 0.371; interaction = 0.972). Significant weight loss was observed among compliers (87.7 kg at baseline vs. 79.7 kg at 12 months; time effect = 0.034); but there was no difference between groups (group effect = 0.299; interaction = 0.996). The mean weight loss at three months was greater in those who completed the 12 months than those who did not (−5.2 vs. -3.2 P = 0.005).

All groups showed improvements in fasting insulin, HOMA-IR and triglycerides (Table 3). Systolic blood pressure, fasting glucose, total cholesterol and leptin levels decreased for the entire study sample, with no difference between groups. There was a significant time by group interaction for self-reported intake of LCn3PUFA (Table 4, p = 0.034), confirmed by a significant group difference in erythrocyte omega-3 index (Table 3, p = 0.009). This effect was primarily due to changes in the Fish + S group, and post hoc analysis revealed a significant difference between the Fish and Fish + S groups (Fish: 3.4 ± 1.0% vs. Fish + S: 4.4 ± 1.6%; p = 0.007).