**2. Material and Methods**

#### *2.1. Animals*

The study was undertaken in accordance with prevailing EU and national guidelines for animal care and welfare and in compliance with ARRIVE guidelines. All animal procedures were approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine Vienna, Austria, and by the Austrian Federal Ministry of Education, Science and Research (permit number BMWFW-68.205/0188-WF/V/3b/2017).

All experiments were performed using 12–14-month-old male C57BL/6 mice. Animals were kept on 24 ◦C with a 12/12-h light/dark cycle and were housed in stable groups of 2–5 mice from the time of weaning. Starting from weaning, the mice were fed either a normal mouse diet (V1124-000, Sniff, Soest, Germany) containing 1.0% calcium, 0.7% phosphorus, and 1000 IU vitamin D/kg, or a CPD containing 2% Ca, 1.25% P, and 20% lactose. The Ca/P ratio of both diets was similar (1.43 for ND and 1.60 for CPD), and both diets had the same energy content (14 MJ/kg) and the same nutrient distribution (carbohydrates, protein, fat). All animals had access to food and tap water ad libitum. Before necropsy, urine was collected overnight in metabolic cages. At necropsy, the mice were exsanguinated from the abdominal vena cava under general anesthesia with ketamine/medetomidine (100/0.25 mg/kg i.p.) for serum collection. Necropsies were performed between 9 a.m. and 3 p.m. for all mice. Some mice maintained on a CPD until 12–14 months of age were switched to ND for two weeks to assess the effect of the diet switch on urinary excretion of catecholamines. Investigators were not blinded to the group allocation of the animals.
