Advanced 3d3 osmometer

Urine samples were analyzed upon delivery to the laboratory and no later than 24 h after the collection. Osmolality was measured in duplicate, by freezing point depression (3D3 Advanced Osmometer, Advanced Instruments, Inc., MA, USA). Specific gravity was measured in duplicate using a handheld clinical refractometer (ATAGO SUR-NE, Tokyo, Japan). UC was determined from an experienced researcher by comparing the color of the urine sample placed in a clear, glass 15-mL tube against white background, under fluorescent lighting, next to an original urine color scale [31 ].

Blood samples were collected from runners following 15 min seated rest and analyzed immediately in duplicate for hematocrit (microhematocrit method) and hemoglobin (cyanmethemoglobin method, Drabkin reagent; Sigma-Aldrich, St. Louis, MO, USA), and the Dill and Costill equation was used to calculate changes in plasma volume [13 (link)]. In the remaining blood, serum and plasma were separated from the blood cells by centrifugation. Aliquots of blood plasma were used fresh for the determination of plasma osmolality by freezing-point depression (3D3 Osmometer; Advanced Instruments Inc., Norwood, MA, USA). The remaining blood plasma and serum were stored frozen (−80 °C) for subsequent analysis of serum glucose and creatine kinase serum sodium and potassium. Glucose and creatine kinase were measured by enzymatic analysis in an automated biochemical analyzer (ACE; Schiapparelli Biosystems Inc., Fairfield, NJ, USA). Electrolytes concentration was measured by selective electrode conductivity in an automated analyzer (Ektachem DT60 II system; Eastman Kodak Co., Rochester, NY, USA). Urine osmolality was measured in duplicate by freezing point depression (3D3 Advanced Osmometer, Advanced Instruments Inc., MA, USA) and specific gravity (USG) with a handheld refractometer (ATAGO SUR-NE, Tokyo, Japan).

A 24-h urine collection was conducted on the last day of each diet. Urine was analyzed for total volume, urinary electrolytes (Easy-Electrolyte Analyzer; Medica, Bedford, MA, USA), and urine osmolality (Advanced 3D3 Osmometer; Advanced Instruments, Norwood, MA, USA). Free water clearance and fractional excretion of sodium and chloride were calculated using standard equations. Urine collections were considered incomplete if collections occurred outside the 20–28 h timeframe, if there were two or more missed collections, or if total volume was less than 500 mL. During the same 24-h period, subjects also wore an ambulatory BP monitor (Oscar 2; SunTech Medical, Morrisville, NC, USA) on their arm. BP was measured every 20 min while the subject was awake and every 30 min during sleep. This device has been validated for brachial BP measurements [20 (link)]. A subject was considered adherent if at least 75% the readings were successful [21 (link)]. Laboratory BP was also measured by an automated oscillometric sphygmomanometer (Dash 2000; GE Medical Systems, Milwaukee, WI, USA) during the experimental visits.