Mussels

The 1-D, pJRES and 2D JRES NMR spectra were converted to an appropriate format for multivariate analysis using custom-written ProMetab software [15 (link)] running within MATLAB (version 7.1; The MathWorks, Natick, MA). All spectra were sectioned into 1960 chemical shift bins between 0.2 and 10.0 ppm, corresponding to a bin width of 0.005 ppm. Note that the 2D JRES spectra were not "binned" along the J coupling dimension at this stage of the processing. Next, a series of bins were removed from each data set: for the canine urine from 4.50–6.45 ppm (residual water and urea); for the mussel adductor muscle from 4.70–5.15 ppm (residual water) and 7.60–7.76 ppm (chloroform); and for fish liver from 4.60–5.20 ppm (residual water). The spectra for each data set were then normalised to a total spectral area of unity for ease of comparison between samples. Next, due to slight pH-induced chemical shift variations of some peaks between samples, groups of bins were each compressed into single bins: for the canine urine ten regions were compressed between 2.40–2.425, 2.52–2.57, 2.66–2.71, 2.935–2.955, 2.96–2.98, 3.105–3.130, 3.72–3.77, 3.955–3.990, 7.08–7.20 and 8.00–8.18 ppm; for the mussel adductor muscle between 7.08–7.10 and 7.84–7.875 ppm; and for fish liver five regions were compressed between 7.74–7.77, 7.77–7.79, 7.94–7.955, 7.97–8.03 and 8.23–8.25 ppm. Compression regions were chosen by visually inspecting the superimposed NMR spectra and then selecting regions of the spectra that showed pH or matrix induced chemical shift variation. Finally, for the fish liver only, the increments of each intact 2D JRES spectrum (i.e. the rows of the 2D data matrix representing each spectrum) were concatenated into a single row vector of dimension 232,448 containing the intensities of each bin in the spectrum, allowing the JRES spectra to be analysed in a similar manner to the 1D and pJRES spectra, described below.

The short-version self-administrated questionnaire developed for the study included questions about the habitual intake of seafood for dinner, as sandwich spread, in salads or as snack meal, with questions focusing on type of seafood, frequency of intake and in some cases portion size. Frequency responses of seafood intake were recorded as: never, less than once per month, one to three times per month, one to two times per week, or three or more times per week. The portion size of seafood for dinner was recorded as follows: half a portion, one portion, one and a half portion, two portions or three portions. One portion corresponds to 150 grams of seafood; e.g. one slice of salmon fillet, three fishcakes or two deciliters of shrimps. The intake of different types of seafood was determined by asking about the intake of 36 different types of lean and fatty seafood (specific fish species, fish as sandwich spread, mollusks, crustaceans and semi-manufactured fish products). In addition it was possible to register intake of seafood not given in the list. The questionnaire also included questions about the use of supplements (cod liver oil, fish oil capsules, multivitamin/mineral mixtures, vitamin B, vitamin D, calcium and iron), in which the product names of the most commonly used supplements in Norway were listed. For the use of bottled or encapsulated cod liver oil, the questionnaire differentiated between the whole year and during winter only. Frequency response was recorded as: one to three times per month, one to three times per week, four to six times per week, or daily. The amount of intake was recorded as a teaspoon (3 mL), child's spoon (5 mL) or tablespoon (10 mL).
In addition, the short FFQ included questions concerning specific food habits, such as frequency of consumption of dairy products (a major contributor to iodine intake), fruits and vegetables, butter and margarine (products fortified with vitamin D (8 μg/100 g) in Norway) and use of fats in cooking. General characteristics like age, weight, height, smoking, physical exercise, medication and interest in eating healthy, were self reported in the questionnaire. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m²). Frequency of exercise was determined by the question: How often do you perform physical exercise for at least 20 minutes (walking, jogging, bicycling, swimming, football, aerobics)? The questionnaire included a short written instruction about habitual intake in addition to our understanding of seafood, which comprise fish, fish products, mussels and crustaceans. On the average it took approximately ten minutes to complete the FFQ.

Total RNA was isolated from 400 μl of plasma from six individuals for whom non-hemolyzed and hemolyzed plasma obtained from different collection tubes but the same blood collection was available. In addition RNA was isolated from 100 μl Ficoll-purified RBCs from the healthy donor and from 400 μl of plasma containing RBC dilution [5 points of a serial dilution series (Kirschner et al., 2011 (link))]. RNA isolation was performed using the mirVana PARIS miRNA isolation Kit (Life Technologies) with small modifications. The RBC samples were mixed with 300 μl cell disruption buffer prior to further processing identical to the plasma samples. Following the denaturing step of the isolation process, 100 μg mussel glycogen (Roche) were added to each sample as carrier to enhance isolation efficiency. After separation and recovery of the aqueous phase, a second phenol–chloroform extraction of the aqueous phase was included to improve the removal of the high protein content of plasma. Following the column-purification part, RNA was eluted using 100 μl ultrapure H₂O (95°C), resulting in a recovery of around 85 μl RNA. For each sample two independent RNA isolations were performed.
For those samples to be used for microRNA profiling 70 μl of the total RNA were further concentrated in order to increase the amount of RNA that could be reverse transcribed. These samples were mixed with 300 mM sodium acetate pH 5.2, 2.5 ng mussel glycogen, and 175 μl 100% Ethanol and incubated over night at −80°C. RNA was precipitated by centrifugation at 17000 g for 20 min at 4°C. Pellets were washed with 1 ml 75% Ethanol and re-precipitated at 17000 g for 10 min at 4°C. After removal of the supernatant RNA was air-dried for approximately 10 min (until the RNA pellet changed color from white to opaque) and then resuspended in 10 μl ultrapure H₂O. RNA concentration was assessed using the Qubit RNA Assay Kit (Life Technologies), however RNA concentrations for the plasma samples were below the limits of detection. All samples were stored at −80°C until further use.

Process waters generated
during various steps of herring processing were collected at Sweden
Pelagic AB in Ellös, Sweden, as follows: (i) RSW from herring
storage on board a boat was collected in March 2020, (ii) salt brines
(13% NaCl) from presalting of herring skin-on fillets or deskinned
fillet pieces were collected in the North Sea (October 2018), and
(iii) marination brines into which the presalted herring is placed
and stored for up to 2 years: (a) salt marination brine (SMB), (b)
spice marination brine (SPB), and (c) vinegar marination brine (VMB).
Mussel (Mytilus edulis) processing
at Vilsund Blue, Nykobing Mors, Denmark, comprises four different
steps: boiling, removal of impurities through treatment with 15% salt
brine, vibration to remove the shells, and a final rinsing step to
remove salt residues. The four different process waters collected
for this study were boiling water (generated during boiling mussels),
juice (generated during dripping of the boiling water from mussels
when transferred on a belt to the next step), salt brine (generated
during brining with salt brine), and rinsing brine (generated while
mussels are rinsed to remove salt). Mussel process waters were sampled
in 2016 and 2017.

Polypeptide profile
of process water samples was investigated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) following the method descried by Laemmli.^{16 (link)} Mini-protean TGX 4–20% precast gels (Bio-Rad
Laboratories, USA) were used to run the electrophoresis. Briefly,
samples containing approximately 20 μg of protein (except for
salt brine from mussel processing (containing 10 μg) and mussel
rinsing water (containing 2 μg)) were mixed with the loading
dye at 1:1 v/v ratio. The protein molecular standard (Bio-Rad Dual
Color, Bio-Rad, USA) ranged between 10 and 250 kDa. Protein bands
were stained by Coomassie Brilliant Blue G-250.