Absolute quantification of urinary neutrophil gelatinase-associated lipocalin by ultra-high-performance liquid chromatography/tandem mass spectrometry and the diagnostic efficacy of acute kidney injury.

RATIONALE: To establish an absolute quantification method for neutrophil gelatinase-associated lipocalin (NGAL) by ultra-high-performance liquid chromatography tandem positive ion electrospray ionization mass spectrometry (UHPLC/MS/MS) and evaluate its diagnostic efficacy for acute kidney injury (AKI). METHODS: Three target peptides of NGA were prescreened by Skyline software, and two of them could be detected in tryptic peptides of NGAL recombinant protein and human urinary NGAL (uNGAL). Peptide (WYVVGLAGNAILR) was then selected as surrogate peptide. The corresponding isotope-labeled peptide as the internal standard was next synthesized. Quantification of uNGAL was based on equations of linear regression, and method validation was then conducted. The diagnostic efficacy of uNGAL for AKI was also evaluated using receiver operating characteristic curve (ROC) analysis. Lastly, the UHPLC/MS/MS and the particle-enhanced turbidimetric immunoassay (PETIA) methods for uNGAL quantification were compared. RESULTS: For the y9 and y10 product ions, the linear regression equations were y = 2.5519x-4.6955 (R2 = 0.994, P<.01) and y = 2.4619x-4.3 (R2 =0.993, P<.01), respectively, and both of the linear ranges were from 0.5 to 15 mg/L. The limits of detection and quantification were 0.037 mg/L and 0.081 mg/L, respectively. The recoveries were from 97.32% to 107.28% at different uNGAL levels, and the within- and between-day CVs for uNGAL quantification were from 0.22% to 7.65% and from 0.66% to 5.97%, respectively. The carryover rates of uNGAL were in the range of 0.70%-0.99%. The area under the ROC curve (AUC) of uNGAL was 0.96 (P<0.01), and the sensitivity and specificity of uNGAL for AKI diagnosis were 90.0% and 92.5%, respectively. In addition, the UHPLC/MS/MS and PETIA methods showed good agreement for uNGAL quantification (y = 0.7112x-0.0139, P = 0.34). CONCLUSIONS: The UHPLC/MS/MS method for uNGAL quantification has a wide linear range, high sensitivity, precision, and recovery, and low carryover rates, and uNGAL detected by this method had high sensitivity and specificity for AKI diagnosis.

Quantification of cystatin-C in human serum by stable isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry.

Cystatin C (CysC)-based estimated glomerular filtration rate is an excellent marker for early diagnosis of Chronic Kidney Disease (CKD). Particle-enhanced nephelometric immunoassay (PENIA) and particle-enhanced turbidimetric immunoassay (PETIA) are commonly used methods for CysC quantification in clinical testing. However, both of them lack of specificity which mass spectrometry offers. In this paper, an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method was established for quantification of CysC in human serum. After CysC denaturation, reduction and alkylation of cysteine residues, trypsin was added for proteolytic digestion of CysC. Specific peptide ALDFAVGEYNK was selected as surrogates for intact CysC protein, then quantified CysC by stable isotope-labelled internal standard peptide A[13C615N]LDFAVGEYNK based on calibration curve method. The calibration curves were y=0.1565x-1.6715 (R2=0.988) and y=1.8785x-2.2497 (R2=0.991) for y9 and y6, respectively. The linear range was 0.1-10mg/L and 0.5-15mg/L for y9 and y6, respectively. The limit of quantification (LOQ) was 0.052mg/L. At different concentrations of CysC, the recoveries were in the range of 80.5%-93.7%, the intraday precisions were in the range of 0.3%-2.2%, and the inter-day precisions were in the range of 0.2%-2.8%. The results show that ID-LC-MS/MS and PETIA methods have higher consistency (y=0.8021x+0.6611, p=0.14), and the mean difference of the two methods was -0.29mg/L, and 95% of the individual difference values were in the range of -0.91mg/L-0.33mg/L.

PAX3 is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells.

Paired box 3 (PAX3) is overexpressed in glioma tissues compared to normal brain tissues, however, the pathogenic role of PAX3 in human glioma cells remains to be elucidated. In this study, we selected the human glioma cell lines U251, U87, SHG-44, and the normal human astrocytes, 1800, which have differential PAX3 expression depending upon the person. SiRNA targeting PAX3 and PAX3 overexpression vectors were transfected into U87 and SHG-44 glioma cell lines, and cell proliferation, invasion, apoptosis, and differentiation were examined by CCK-8 assays, transwell chamber assays, tunnel staining, Annexin V/PI analysis, and Western blotting, respectively. In addition, we used subcutaneous tumor models to study the effect of PAX3 on the growth of glioma cells in vivo. We found that PAX3 was upregulated in the three glioma cell lines. PAX3 knockdown inhibited cell proliferation and invasion, and induced apoptosis in the U87MG glioblastoma cell line, whereas PAX3 upregulation promoted proliferation, inhibited apoptosis, and increased invasion in the SHG-44 glioma cell line. Moreover, we found that targeting PAX3 expression in glioma cell lines together with chemotherapeutic treatment could increase glioma cell susceptibility to the drug. In subcutaneous tumor models in nude mice using glioma cell lines U-87MG and SHG-44, inhibition of PAX3 expression in glioblastoma U-87MG cells suppressed tumorigenicity, and upregulation of PAX3 expression in glioma SHG-44 cells promoted tumor formation in vivo. These results indicate that PAX3 in glioma is essential for gliomagenesis; thus, targeting PAX3 or its downstream targets may lead to novel therapies for this disease.