Pharmacokinetic study protocol

The aspects of the protocol concerning animal welfare were approved by RTC animal-welfare body. The animal treatment phase for this study was performed at the European Research Biology Center S.R.L (ERBC) facilities, former Research Toxicology Center (RTC), Pomezia, Rome, Italy. Procedures and facilities were compliant with the requirements of the Directive 2010/63/EU on the protection of animals used for scientific purposes.

Sprague Dawley rats, 7–9 weeks old and with body weight of approximately 170–200 g, were ordered from Envigo RMS srl, San Pietro al Natisone, Udine, Italy. The animals were housed in a limited access rodent facility for an acclimatisation period of approximately 4 weeks before the start of treatment. Animal room controls were set to maintain temperature and relative humidity at 22 ± 2 °C and 55% ± 15%, respectively. Drinking water was supplied ad libitum to each cage via water bottles. Commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Milan, Italy) was offered ad libitum throughout the study, except for an overnight fast prior to dosing and approximately 2 h after dosing. SAM was administered in fasting conditions to avoid possible interference of food consumption on SAM intestinal absorption, SAM absorption is higher in fasting conditions (Cameron et al. 2020). For this reason, rats were deprived of food the night before supplement administration, fasting was extended to the two hours following administration, when most of the absorption occurs.

The pharmacokinetic profile of SAM phytate, compared to SAM PTS, was investigated in rats after single oral administration, according to the following experimental design:

Group 1 comprised 16 male and 16 female rats that were given 133.7 mg/kg of SAM phytate (test substance);

Group 2 comprised 16 male and 16 female rats that were given 95.4 mg/kg of SAM PTS (reference substance).

Test and reference substances were dissolved in water and administered by gavage to each animal at a dose volume of 10 mL/kg. Since the substances were dissolved in water, no control group was deemed necessary. Rats were administered with equimolar solutions of SAM PTS and SAM phytate (125 μmol/kg of body weight). The following investigations were performed: clinical signs, body weight and blood sampling for pharmacokinetic profile, which was carried out as follows: pre-dose and 0.5, 1, 1.5, 2, 4, 8, and 24 h after dosing. At each sampling time, at least 0.8 mL of blood samples were collected from the tail vein of 4 males and 4 females per each sampling time. Each animal was sampled at a maximum of two alternating time points. Maximal care was taken to avoid haemolysis of blood samples. Samples were transferred into tubes containing EDTA anticoagulant and centrifuged at room temperature. The plasma was divided into two aliquots of 200 µL each. Samples were stored at − 80 °C until analysis. All samples were analyzed within 2 months.

Animals that had completed the scheduled test period were killed with carbon dioxide at the end of the last blood sampling procedure. No necropsy was performed and no organs were retained.