Background and objectives: The authors have previously shown that urine neutrophil gelatinase-associated lipocalin (NGAL), measured by a research ELISA, is an early predictive biomarker of acute kidney injury (AKI) after cardiopulmonary bypass (CPB). In this study, whether an NGAL immunoassay developed for a standardized clinical platform (ARCHITECT analyzer, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL) can predict AKI after CPB was tested.Design, setting, participants, & measurements: In a pilot study with 136 urine samples (NGAL range, 0.3 to 815 ng/ml) and 6 calibration standards (NGAL range, 0 to 1000 ng/ml), NGAL measurements by research ELISA and by the ARCHITECT assay were highly correlated (r ؍ 0.99). In a subsequent study, 196 children undergoing CPB were prospectively enrolled and serial urine NGAL measurements obtained by ARCHITECT assay. The primary outcome was AKI, defined as a >50% increase in serum creatinine.Results: AKI developed in 99 patients (51%), but the diagnosis using serum creatinine was delayed by 2 to 3 d after CPB. In contrast, mean urine NGAL levels increased 15-fold within 2 h and by 25-fold at 4 and 6 h after CPB. For the 2-h urine NGAL measurement, the area under the curve was 0.95, sensitivity was 0.82, and the specificity was 0.90 for prediction of AKI using a cutoff value of 100 ng/ml. The 2-h urine NGAL levels correlated with severity and duration of AKI, length of stay, dialysis requirement, and death.Conclusions: Accurate measurements of urine NGAL are obtained using the ARCHITECT platform. Urine NGAL is an early predictive biomarker of AKI severity after CPB.
Elevated levels of Neutrophil Gelatinase Associated Lipocalin (NGAL) in the urine have been established as a promising diagnostic indicator for acute kidney injury (AKI). Development of a quantitative diagnostic immunoassay for NGAL in urine prompts an understanding of the native protein analyte and its structural similarity to the selected recombinant protein used to calibrate the assay. Here we present findings from various analyses on the native human NGAL protein in urine (huNGAL). Chromatographic size‐fractionation of individual human urine specimens indicate that the predominant form of huNGAL is monomeric, although oligomeric forms are detected in some specimens. When observed, NGAL‐containing oligomers are shown to occur through inter‐protein disulfide linkage. An enriched huNGAL protein sample from a pool of urine specimens was analyzed by two‐dimensional electrophoresis (2DE) with Western Blotting to elucidate size and charge distribution of NGAL‐active isoforms. Although all identified huNGAL isoforms displayed molecular weights near the predicted intact polypeptide sequence, the charge distribution of the isoforms was surprisingly wide (pI 5.9‐9.1) given a theoretical pI of 9.0 for the native polypeptide. Structural properties of the enriched huNGAL are compared to those of the immunoassay calibrator protein as well as NGAL protein from other recombinant and native sources.
A simple spectrophotometric method for the determination of citrate in urine is described. This method is based on the complexation of Cu(II) with the Citrate at an elevated pH, which results in the formation of blue colored bis-citrate-Cu(II) complex, measured at 760 nm. The method is applied to the determination of low level of citrate in the urine sample. The relative standard deviation (RSD) was more than 1.5% in the range investigated. The urinary citrate level in 100 stone formers had a median value of 0.78 mM while in the control group the median value of citrate has been 1.3 mM.
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