Effect of high intake of cod or salmon on serum total neopterin concentration: a randomised clinical trial

The study design, the description of study participants, the study setting and the protocol for study visits have been published in detail previously [19]. In brief, the study population consisted of adults with overweight or obesity, and all participants were of Norwegian ethnic origin (Caucasian) living in the Bergen area in South-Western Norway. Inclusion criteria were; BMI ≥ 27 kg/m², fasting blood glucose ≤ 7·0 mmol/l and age 18–69 years. Exclusion criteria were high habitual fish/seafood intake (> 500 g/week), pregnancy, incompatibility with fish consumption (allergies, intolerance and/or dislike), diagnosed diabetes mellitus, heart disease or gastrointestinal diseases, use of medications affecting lipid metabolism or glucose homoeostasis, use of anti-inflammatory medications, use of supplements containing n-3 PUFAs, intentional weight loss and large fluctuation in body weight (> 3 kg) over the previous 2 months. Participants were interviewed about their fish/seafood intake before they were included in the study, and those with a regular fish intake > 1 fish dinner per week were instructed to avoid eating fish for 4 weeks before the baseline visit. Very few participants (< 10%) had an intake above 1 fish dinner per week before enrolment to the study and went through a 4-week fish-free period before the start of the intervention period.

The study was designed as a randomised, controlled intervention study with a parallel group design, with three intervention arms: Atlantic cod (wild-caught Gadus morhua) in weekly doses of 750 g, Atlantic salmon (Norwegian farmed Salmo salar) in weekly doses of 750 g, and a no-fish group as the Control group. The intervention period was 8 weeks. In all, 76 participants were included in the study and were randomly assigned to the Cod group (N = 27), Salmon group (N = 27) or the Control group (N = 22). The participants were randomised into the different groups by the project manager by drawing lots, and the participants were informed about their group allocation during the baseline visit. All examinations were conducted at the Clinical Research Unit at the Haukeland University Hospital, Bergen, Norway. To enhance compliance, the participants were contacted by phone approximately 1 week prior to baseline and end point visits, during which they were informed of the schedule and procedures for the following visit. Also, a text message was sent 1–3 days before the 8 week visit, as a reminder of how to prepare for the upcoming visit. For any inquires during the trial period, members of the research group could be reached by email or telephone. Compliance was monitored through interviews; after 1, 4 and 8 weeks of the intervention period, the participants in the fish eating groups were asked how many dinners with cod/salmon they had not eaten since last contact, instead of asking how well they had complied, to make it less dissuasive for the participants to report missing intake. Noncompliance was defined as not following the protocol in regard to fish intake (omitting more than three fish dinners in the fish eating groups during the intervention period), other dietary changes or use of prescription medicine not compatible with the inclusion criteria, or changes in physical activity. As reward for completing the study, participants were offered a dietary consultation with a student dietician at the last visit and all the results of analyses of their blood samples.

The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures were approved by the Regional Committee for Medical and Health Research Ethics of Western Norway (REC no.: 2011/572). Written informed consent was obtained from all subjects.

Health professionals performing blood sampling, and personnel conducting the laboratory analyses, were all blinded to participants’ identity and group allocation. All data were analysed anonymously. This trial was registered at clinicaltrials.gov as NCT02350595.

Cod and salmon fillets were provided to the participants as frozen-skin and boneless-fillet portions (mean weight with standard deviation; 150 (SD 10) g; Lerøy Seafood Group ASA). Pallets of fish were chosen at random from Lerøy’s warehouse in Bergen, Norway. The cod and salmon fillets were supplied free of charge to the participants, and were distributed at the baseline visit or at any time during the study period, if preferred. Participants in the Cod group were instructed to eat five dinners per week containing 150 g of cod fillet, and participants in the Salmon group were instructed to eat five dinners per week containing 150 g of salmon fillet. The participants in the fish-eating groups were told not to exceed a total amount of 750 g of fish/week, not to consume any other types of fish or seafood during the study period, and to maintain their normal eating habits throughout the study period apart from eating the mandatory amount of 750 g fish/week. The Control group was also instructed to continue their normal eating habits, except to avoid fish and seafood intake. Participants in both fish intervention groups received a booklet with dinner recipes for inspiration and to help them to increase the variation of their meals, as previously described [22]. The participants in the Control group did not receive any recipes, to avoid them being inspired to change their dietary habits during the study period.

Subjects in all groups were instructed not to change their physical activity level during the intervention period. The participants’ habitual lifestyle were recorded at the baseline and the endpoint visits, using a questionnaire for reporting physical activity. The participants were asked about the types of physical activity they engaged in, such as whether they worked out in a gym, were members of sports clubs or whether they worked out individually, the type of physical activity (e.g. hiking, running, biking) and the number of hours of light physical activity (not sweaty/not breathless) or hard physical activity (with sweat/breathless). The participants completed the questionnaire at the baseline and endpoint visits. The weekly number of hours and the intensities of the physical activities were coded as continuous variables, and reported physical activity were similar between the groups at baseline and were not changed within the groups during the study period [19].

The total study period was 8 weeks, with baseline visits between August 22, 2011 and September 19, 2011. Examinations were conducted in the morning after an overnight fast. The subjects were instructed not to eat or drink anything except water, and not to use substances containing nicotine after 22.00 h the previous day, and to avoid physical exercise and alcohol for 24 h before each sampling day.

Body height was measured at the baseline visit, using a wall-mounted stadiometer (Seca 222; Seca). Body weight and body composition were measured using a bioelectrical impedance analysis device (InBody 720; Biospace Co. Ltd) at the baseline and endpoint visits.

Fasting blood samples were collected at baseline and after 8 weeks, in BD Vacutainer SST II Advance gel tubes (Becton, Dickinson and Company) for isolation of serum. The staff complied with a strict protocol for pre-analytical sample handling to ensure high sample quality. Blood samples were centrifuged after 30 min at room temperature, and serum were immediately aliquoted and frozen at − 80 °C until analyses. Participants provided morning urine at both visits for measurements of markers of kidney function. Urine samples were immediately aliquoted and frozen at − 80 °C upon participants’ arrival to the hospital.

Participants completed dietary records of the 5 preceding days before the baseline visit and the 5 preceding days before the 8-week visit, including at least 1 weekend-day. The intakes of energy, protein, fat, saturated fatty acids (SFA), cis-monounsaturated fatty acids (cis-MUFA), cis-polyunsaturated fatty acids (cis-PUFA), carbohydrates, iron, magnesium, selenium and zinc were calculated from the participants’ dietary records using the ‘Mat på Data 5.1’ software [23], which contains information on the nutrient contents in food items sold in Norway. This food database does not contain information of copper contents in foods. Food records were checked for completeness at both study visits.

All serum/urine samples for each analysis were analysed pairwise for each participant in random order on the same day, and samples were not thawed previously. Total neopterin was measured in serum after precipitation of proteins by trichloroacetic acid, which oxidises 7,8-dihydroneopterin to neopterin, and total neopterin was analysed by Bevital AS (Bergen, Norway, http://​www.​bevital.​no) using liquid chromatography combined with tandem mass spectrometry, as previously described [24]. Vitamin A (all-trans retinol) and vitamin E (α-tocopherol and γ-tocopherol) were measured by liquid chromatography-tandem mass spectrometry [25] by Bevital AS. Serum magnesium and iron were analysed with the Cobas c111 system (Roche Diagnostics GmbH, Marburg, Germany) using the MG (Magnesium) and IRON2 (Iron Gen.2) kits for Cobas c111 (Roche Diagnostics GmbH). Copper and selenium in serum were quantified by the Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, using an Elan DRC-e Inductively Coupled Plasma Mass Spectrometer (Perkin Elmer MDS Sciex, Concord, ON, Canada) in standard mode, after dilution 1:15 with 1% Triton^® X-100 and 0.33% w/v pro analysis HNO₃.

Urine concentrations of cystatin C and T-cell immunoglobulin mucin-1 (TIM-1) were measured as markers of kidney function, using the Human Cystatin C Quantikine^® ELISA (DSCTC0) and the Human Urinary TIM-1/KIM-1/HAVCR Quantikine^® ELISA (DKM100) from R&D Systems (Bio-Techne). Creatinine in urine was quantified using the CREP2 (Creatinine plus ver.2) kit from Roche Diagnostics using the Cobas c111 system (Roche Diagnostics GmbH).

The sample size calculation for this trial was originally conducted with the aim to investigate the effects of high intake of cod or salmon on postprandial glucose regulation after a standardised breakfast in participants who were overweight or obese [19]. We have previously presented an estimate that it was necessary to include 76 participants divided into 3 groups to ensure that 20 participants in each group completed the trial with satisfactory compliance, with a power of 80% and α of 0.05, and of these, 65 participants were included in statistical analyses [19]. In the present study, we wanted to further explore serum and urine samples from this randomised clinical trial to investigate the effect of a high intake of cod or salmon on serum concentrations of total neopterin. This is the first study to investigate the effects of 8 weeks of high intake (750 g/week) of cod or salmon on serum total neopterin concentration in adults who were overweight or obese, therefore, no data are available for sample size calculation for the present study.

Statistical analyses were conducted using SPSS Statistics 25 (SPSS, Inc., IBM Company). Subjects who did not complete the study were excluded from the statistical analyses. For analytes in serum and for estimated intake of micronutrients, most data were not normally distributed according to the Shapiro–Wilk test, therefore, the nonparametric Wilcoxon’s signed-ranks test was used to investigate changes within groups. The Kruskal–Wallis test was used to compare values between the three groups at baseline. Changes within the groups were compared using the Kruskal–Wallis test followed by group comparisons adjusted using the Bonferroni correction for multiple tests whenever between-group differences were detected. All statistical testing within and between the groups were unadjusted. Correlations between parameters were tested using two-tailed Spearman’s correlations test. Data are expressed as medians and 25th, 75th percentiles. Categorical data were compared using the Pearson’s χ² test. All comparisons were two-sided, and p < 0.05 was considered statistically significant.

In total, 76 participants were included in the study and completed the first study visit, and 68 participants completed the trial. One participant (a woman in the Salmon group) was excluded from statistical analysis after analyses of postprandial blood glucose revealed she had prediabetes, and two participants (one woman in the Cod group and one man in the Salmon group) were withdrawn from analysis because they did not comply with the protocol. From two of the participants (one man in the Salmon group and one man in the Control group), we did not have a sufficient amount of blood serum for analyses, and these two subjects were excluded from all analyses in the present paper. In total, 63 subjects (27 men and 36 women) were included in the statistical analyses. The flow of participants in the study is presented in Fig. 1.

Groups were similar at baseline in regard to gender distribution, age, BMI, percentage body fat or percentage muscle mass (Table 1), with median age 45.6 (25th,75th percentile 37.1,53.9) years and median BMI 32.3 (25th,75th percentile 29.6,35.7) kg/m². After 8 weeks, no changes were seen in any of the groups for BMI or percentage body fat or muscle (data not presented).

The estimated median daily intakes of energy, protein, fat, SFA, cis-MUFA, cis-PUFA, carbohydrates, iron, magnesium, selenium and zinc were calculated from 5-day food diaries prior to baseline and endpoint visits, and intakes were similar between the groups at baseline. Participants reported no intake of seafood (beside the mandatory cod/salmon intake in the fish eating groups), liver or kidney in the food diaries. The selenium intake was similar in all groups, but tended to be increased in the Cod group (P = 0.052) and in the Salmon group (P = 0.086) from baseline to endpoint when compared to the Control group, with no difference between the Cod and the Salmon group (Table 2). The zinc intake was significantly reduced in the Cod group and unchanged in the Salmon group from baseline to endpoint when compared to the Control group, with no difference between the Cod group and the Salmon group. The intakes of energy, protein, fat, SFA, cis-MUFA, cis-PUFA, carbohydrates, iron and magnesium were not changed within any of the groups from baseline to 8 weeks.

Serum concentrations of total neopterin, copper, iron, magnesium and selenium were similar between the groups at baseline. Total neopterin serum concentration was significantly reduced from baseline to endpoint in the Cod group when compared to the Control group, but this reduction was not significantly different when compared to the Salmon group, and the changes within the Salmon group and the Control group were not significantly different (Table 3, Fig. 2).

The serum concentration of selenium was significantly increased in the Cod group when compared to the Control group, but was not significantly different from the Salmon group. The change in neopterin concentration from baseline to endpoint was similar in the Salmon group and the Control group. The magnesium serum concentration was reduced after 8 weeks in the Cod group, but this change was not significantly different from the changes in the Salmon group and the Control group. In the Control group, serum concentration of iron was reduced from baseline to endpoint, but this change was not significantly different from the fish eating groups. Serum concentrations of copper (Table 3) and all-trans retinol, α-tocopherol and γ-tocopherol [20] were not changed within or between the groups.

At baseline, serum concentration of total neopterin was not correlated with serum concentrations of copper, iron, magnesium, selenium, all-trans retinol, α-tocopherol and γ-tocopherol (Table 4). At endpoint, when all groups were considered together, serum total neopterin concentration was positively correlated with magnesium concentration (r = 0.368, P = 3.5 × 10^–3), but was not correlated with the concentrations of the other elements or vitamins measured in serum (Table 5). When the experimental groups were investigated separately, no statistically significant correlations between serum total neopterin concentration and concentrations of elements and vitamins were seen within any of the groups (data not presented).

Cystatin C and TIM-1 were measured in urine sampled at baseline and endpoint as measures of kidney function since serum neopterin concentration is increased in patients with kidney dysfunction [26, 27]. We found no significant correlations between serum total neopterin concentration and urine concentrations (relative to creatinine) of cystatin C and TIM-1 at baseline and endpoint (P > 0.4, data not presented).