Urine analyte analysis
Urine protein concentrations were measured using commercially available multiplex human cytokine assays and a Bioplex multiplex bead array reader from Bio-Rad Laboratories Inc, (Hercules, CA) that uses a Luminex 100 system (Luminex Corp., Austin, TX). As per manufacturer’s suggestion, a diluent containing PBS (pH 7.4) and 0.5% BSA was used to prepare the standards. Prior to analysis, the bead array reader was calibrated per manufacturer’s instructions. Urine proteins from premixed kits (Bio-Rad) analyzed were IL6, IL8, monocyte-chemoattractant factor-1 (MCP1), macrophage inhibitory protein (MIP1)α and tumor necrosis factor (TNF)α. All results were expressed as means of four replicate values. Limits of quantification were defined as the lowest and highest concentration at which the percent coefficient of variation were below 10%. The interpolated lower limits of quantification for IL6, IL8, MCP1, MIP1α, and TNFα were 1, 9, 13, 8, and 8 pg/ml respectively.
Recovery of proteins spiked into urine samples
Four urine samples with a large degree of variation in typical measurable components were chosen as test samples (Na, <10 to 106 mM; K, 8 to 44 mM; Ca, <2 to 6.5 mg/dl; UUN, <50 to 1406; Osm, 252 to 629 mOsm; pH, 4.6 to 7.8; measured by the Medical University of South Carolina clinical laboratory; Supplemental Table 2
). Each urine sample was spiked with a known quantity of the protein standards within the measurable range of the standard curve (IL6, 1046; IL8, 588; MCP1, 679; MIP1α, 567; TNFα 2294 pg/ml). Each analyte and each unspiked and spiked urine sample were assayed in quadruplicate and averaged.
Recovery of proteins after a series of dilutions with standard diluent
A series of dilutions of the four urine samples used in experiment 1 were made by diluting with the diluent used to create the standard curve (PBS/0.5% BSA). Samples diluted 1:20, 1:10, 1:5, and neat were analyzed in quadruplicate and averaged. Then, samples first diluted 1:20, 1:10, 1:5, 1:2 and neat were spiked with standard (IL6, 860; IL8, 693; MCP1, 687; MIP1α, 424; TNFα, 2057 pg/ml) and analyzed in quadruplicate and averaged.
Standard addition for determining protein concentrations
An experiment was designed using standard addition as previously described7
to determine the unknown concentrations of proteins. Urines 1, 2 and 3 were spiked with five levels of standard (2743 to 7 pg/ml), and these and an unspiked sample were analyzed in quadruplicate. Regression lines for the points generated for each urine and each analyte (added concentration of standard— x-axis; instrument response—y-axis) were created using the resistant least trimmed squares method (function ltsreg in the MASS package)8