1 electrolyte analyzer

Urine from each individual metabolic cage drained over a mesh wire feces trap and into clean 50 mL conical centrifuge tubes. It was collected in 24 h periods over 19 days; every day at 12:00 h. Metabolic cage pans were replaced with clean bottoms daily to reduce contamination of urine samples from minuscule amounts of food and/or feces. Urine volumes were recorded daily. Urine samples were transferred to Eppendorf tubes, spun at 10K rpm for 5 min to remove particulate contamination and sediment, and placed in a −80 freezer. Daily sodium excretion values were calculated from 24 h urine volumes and sodium concentrations measured by a Nova Biomedical 1 electrolyte analyzer (Waltham, MA). Total cumulative sodium balance was calculated by summing the sequential daily differences between intake and total sodium excretion and expressed in mEq ± SEM over a period of 19 days (Beierwaltes et al. 1982).

Representative fecal samples were collected from all five groups to determine fecal sodium excretion as a percent of total sodium excretion (urinary and fecal sodium excretion). n = 3 for C, F, F + HS, and HS groups, and n = 4 for the G + HS group. Individual 24 h fecal samples were transferred to 50 mL conical centrifuge tubes and placed in a −80 freezer until further analysis. Fecal samples were thawed to room temperature, allowed to air dry, and total fecal dry weight was recorded. One gram of feces was combined with 4 mL of distilled water and homogenized with a Tissuemiser (Fisher Scientific, Pittsburgh, PA). The fecal slurry was transferred to a 1.5 mL eppendorf, spun down at 1000 g at 4°C for 5 min, the supernatant pipetted into a new eppendorf. Daily fecal sodium excretions were calculated from sodium concentrations using a Nova Biomedical 1 Electrolyte Analyzer (Waltham, MA) and total fecal mass to achieve a 24 hr fecal sodium excretion and fecal excretion as a percent of total sodium excretion (urinary plus fecal sodium excretion) (Beierwaltes et al. 1982).