*2.4. Measurements*

Anthropometric measurements included body mass and height. Body mass was assessed using a calibrated weighing scale with an accuracy of ± 0.1 kg and body height was assessed using a stadiometer with an accuracy of ± 0.5 cm. The measurements were conducted by a professional nutritionist, according to the recommended procedure [35]. Afterward, the BMI was calculated, based on the Quetelet equation and interpreted to verify the exclusion criteria [30].

Vitamin D status was assessed based on the total serum 25(OH)D level at baseline (t0), after 4 weeks of dietary intervention (t4), and after 8 weeks of intervention (t8). For the analysis, venous blood samples were drawn by a qualified nurse in a certified medical analysis laboratory in Warsaw, Poland; participants did not have to be in a fasting state before blood collection. Total serum 25(OH)D level tests were performed on BS Mindray BS-200 Chemistry Analyzer using Diazyme EZ Vitamin D assay and the dual vial liquid stable (latex enhanced immunoturbidimetric) method, which enabled determination of total 25(OH)D in the range of 19.0–369.5.8 nmol/L. For this method, a comparison of the EZ Vitamin D assay (y) using samples measured with LC–MS (liquid chromatography–mass spectrometry)/MS in the validation gave the following correlation: y = 1.0297x−0.813 for R<sup>2</sup> = 0.9622 and a comparison of the EZ Vitamin D assay using samples measured with a commercially available 25(OH)D immunoassay in the validation gave the following correlation: y = 1.1537x−1.2321 for R<sup>2</sup> = 0.9716. The assay precision for the method is defined by percent coe fficient of variation (%CV) lower than 5% at 75 nmol/L. The method is certified by the Centers for Disease Control and Prevention within the Vitamin D Standardization–Certification Program (CDC VDSCP) and meets the performance target set by the Vitamin D External Quality Assessment Scheme (DEQAS) advisory panel.

Each sample was assessed by the same person, in the same conditions, with the same equipment, and using exactly the same methodology, for each sample within 1 hour from drowing the blood samples. The obtained results of total serum 25(OH)D level were compared to the following reference values: <50 nmol/L—inadequate, 50–250 nmol/L—adequate, >250 nmol/L—potentially toxic [11,25,36,37].

In order to provide the necessary safety precautions, during the whole experiment, participants had their vitamin D intake and blood pressure controlled. As the intervention comprised additional intake of vitamin D, the total vitamin D intake was controlled throughout the experiment, using the Vitamin D Estimation Only—Food Frequency Questionnaire (VIDEO-FFQ), which was previously validated in a group of Polish women aged 20–30 years [38]. Afterward, the obtained vitamin D intake was compared with the upper intake level (UL) of 100 μg [14]. At the same time, because of high salt content in smoked salmon (1.5 g/50 g), the blood pressure of participants was controlled throughout the experiment (once a week), using Omron Healthcare BP 710N blood pressure monitor, according to the recommended procedure [39]. The observed systolic and diastolic blood pressure values were compared with the standard reference values of 140 mmHg and 90 mmHg, respectively, and the increase of blood pressure above the recommended values observed during two following weeks was decided to be interpreted as a reason to suspend participation in the experiment. Neither for vitamin D intake, nor for blood pressure were the excessive values stated, so it was interpreted as obtaining the required safety of dietary intervention.
