2.3. Serum Metals and Ceruloplasmin Ferroxidase Activity

Fasting serum (30-min clotting time) was collected between 07:00 and 08:00 following subject check-in during both the baseline and intervention inpatient phase. Serum and sweetened beverage aliquots were digested in 1N Ultrex II nitric acid (Avantor JT Baker, Radnor Township, PA, USA) at 4 °C for 24 h then centrifuged at 3000 rpm/4 °C for 12 min in a Sorvall Legend X1R centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) and supernatant collected. Iron, copper, and zinc levels were measured by inductively coupled plasma mass spectrometry at the UC Davis Interdisciplinary Center for Plasma Mass Spectrometry. Sweetened beverage samples were analyzed separately to determine if metal content in the Kool-aid drink mixes were significantly different from one another; no significant differences were found (Figure S1). Ceruloplasmin ferroxidase activity was measured with a colorimetric assay (EIACPLC, Invitrogen, Carlsbad, CA, USA). According to the manufacturer’s protocols, samples were diluted 1:50 in the commercial assay buffer and a colorimetric ceruloplasmin substrate was added. Plates were incubated at 30 °C for 60 min and absorbances were read at 560 nm using a Synergy H1 microplate reader (Bio Tek, Winooski, VT, USA).