Procedures

Procedure 1: Analysis of milk minerals

Equipment, software, and supplies

• Vortex
• Heat blocks
• Inductively coupled plasma-optical emission spectroscopy (Spectro, CIROS ICP Model FCE12, Kleve, Germany)

Reagents and solutions

• Nitric acid
• Hydrogen peroxide
• Tomato leaf standards (National Institute of Standards and Technology, SRM 1573A, Gaithersburg, MD, USA)

Environmental Conditions

Holding Area and/or Sample Collection Area

Anesthesia: Yes, not specified

Steps

1. Study animals, animal husbandry, and experimental design are described in a previous publication (Hadsell et al. 2012).
2. Milk is collected into pre-weighed vials from anesthetized dams (second lactation) at 9 days postpartum under a gentle vacuum and flash frozen.
3. Milk samples are thawed at 37 °C and mixed by vortexing.
4. A 0.2 mL sample is taken and weighed before transferring to a 100 mL glass digestion tube where samples are pre-digested overnight with 3 mL of ultra-pure concentrated nitric acid.
5. Samples are heated to 125 °C on a digestion block for a minimum of 2.5h (with refluxing), until all the orange smoke dissipates from the tubes.
6. Hydrogen peroxide (2 mL) is added to each tube and incubated at 125 °C for 1h. This step is repeated a second time.
7. Samples are brought to dryness at 200 °C and then suspended in 10 mL of 2% nitric acid.
8. Elemental analysis is carried out using inductively coupled plasma-optical emission spectroscopy. Certified standards are used to calibrate the instrument daily (tomato leaf standards).
9. Elements are measured using the following emission lines: Ca (219.770 nm), Cu (327.390 nm), Fe (262.820 nm), K (766.490 nm), Mg (294.200 nm), Na (589.592 nm), P (253.565 nm), S (182.034 nm), and Zn (206.190 nm)