Biospecimen collection protocols for any given participant in the IDATA study were determined by assigned Group. Groups 1 and 3 had their first study center visit at M0, while Groups 2 and 4 had theirs at M6. Subsequently, the second study center visits were at M6 and M11 respectively. For more information on the Group assignments and study center visits, see the Study Summary.

Blood was drawn from all participants at both study center visits. Two large vials of sodium heparin treated blood were collected. Off of the first, one 1.0 mL vial of whole blood was aliquoted off. Then the vials were centrifuged and ultimately divided into f our 1.8 mL of plasma, two 1.8 mL vials of red blood cells and two 1.8 mL vials of buffy coat. Serum was also collected and divided into two 1.8 mL vials and one vial with 0.25 mL serum and 1 mL 6% metaphosphoric acid (MPA). The collection was kept at 2-8°C, until centrifuged within two hours for 15 minutes at 3500 rpm, and ultimately stored at -70°C.

Participants were asked to collect two first morning void and 24-hour urines, six months apart, to measure the concentration of nitrogen, sodium, and potassium, which are reference biomarkers for protein, sodium, and potassium intakes, respectively. At the end of study center visits at months 1, 6, or 12, depending on study group, a 24-urine collection kit that included containers, instructions, and a cooler were given to participants to take home. On urine collection days, which occurred approximately 7-10 days after a study center visit, participants collected 100mL of the first void (FMV) of the morning, and after that began collecting urine for the next 24-hours, including the first void of the next morning. They were also asked to take a 100mg para-aminobenzoic acid (PABA) tablet, a marker for completeness of 24-hour urine collection, at breakfast and dinner. Participants were given a urine collection log to report any missed voids and time at which PABA was taken. Because the OPEN study reported that determination of completeness of 24-hour urine collections by either PABA or self-reported missing voids had no effect on results, the PABA analyses were not conducted.

In addition to collecting urine samples, participants were also asked to collect two saliva samples on the same day as the urine collection. Participants were asked to provide a saliva sample upon waking prior to eating, drinking, smoking or brushing teeth. In the evening, another sample was to be collected at least 30 minutes after eating, drinking, smoking or brushing teeth. If this was not feasible they were asked to rinse thoroughly with water in the mouth at least 5 minutes prior to collecting the sample. Participants were given a saliva collection log to record time of collection along with any antibiotic use that day.

The 24-hour urine and saliva collections were delivered to the study center by a courier. The urine was weighed, aliquoted into 5mL cryovials, and stored at -70°C until being sent to the biorepository. The FMV was aliquoted into one 4.5 mL vial and five 1.8 mL vials. The 24-hour urine collection was aliquoted into five 1.8 mL vials and five 4.5 mL vials. The saliva collections were each aliquoted into two 1.8 mL vials.

Number of Biospecimen Vials Collected Per Person

Material	1st Collection	2nd Collection
Plasma (1.8 mL)	4	4
Whole Blood* (1.0 mL)	1	1
RBC (1.8 mL)	2	2
Buffy Coat* (1.8 mL)	2	2
Serum (1.8 mL)	2	2
Serum MPA (0.25 + 1.0 mL)	1	1
FMV (4.5 mL)	1	1
FMV (1.8 mL)	5	5
24 Hour Urine (4.5 mL)	5	5
24 Hour Urine (1.8 mL)	5	5
Saliva am* (1.8 mL)	2	2
Saliva pm* (1.8 mL)	2	2

*Note: Consent constraints do not allow human DNA to be assessed.