Urea concentration determinations

Urea nitrogen in BALF samples was determined using Abcam 96-well colorimetric urea assay (catalog no. Ab83362) as directed by kit instructions. A 25-μl volume of BALF sample was used for analysis. Standards used in the assay ranged from 1 to 5 nmol per well. The kit directions indicate that the lower limit of detection is 0.5 nmol per well.

Urea nitrogen in serum samples was determined at Charles River Laboratories (CRL)–Mattawan using an automated Beckman Coulter AU5800 chemistry analyzer as directed by the product insert (Beckman Coulter OSR6134, OSR6234, or OSR6634). In this method, urea was hydrolyzed enzymatically by urease to yield ammonia and carbon dioxide. The ammonia and α-oxoglutarate were converted to glutamate in reaction catalyzed by l-glutamate dehydrogenase. At the same time, a molar equivalent of reduced nicotinamide adenine dinucleotide (NADH) was oxidized. Two molecules of NADH were oxidized for each molecule of urea hydrolyzed. The rate of change in absorbance at 340 nm was directly proportional to the blood urea nitrogen concentration in the sample. Serum urea nitrogen was linear from 2 to 130 mg/dl.