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1.
Clin Chem ; 65(3): 406-418, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30647123

RESUMO

BACKGROUND: Clinical practice guidelines recommend estimation of glomerular filtration rate (eGFR) using validated equations based on serum creatinine (eGFRcr), cystatin C (eGFRcys), or both (eGFRcr-cys). However, when compared with the measured GFR (mGFR), only eGFRcr-cys meets recommended performance standards. Our goal was to develop a more accurate eGFR method using a panel of metabolites without creatinine, cystatin C, or demographic variables. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry assay for acetylthreonine, phenylacetylglutamine, pseudouridine, and tryptophan was developed, and a 20-day, multiinstrument analytical validation was conducted. The assay was tested in 2424 participants with mGFR data from 4 independent research studies. A new GFR equation (eGFRmet) was developed in a random subset (n = 1615) and evaluated in the remaining participants (n = 809). Performance was assessed as the frequency of large errors [estimates that differed from mGFR by at least 30% (1 - P30); goal <10%]. RESULTS: The assay had a mean imprecision (≤10% intraassay, ≤6.9% interassay), linearity over the quantitative range (r 2 > 0.98), and analyte recovery (98.5%-113%). There was no carryover, no interferences observed, and analyte stability was established. In addition, 1 - P30 in the validation set for eGFRmet (10.0%) was more accurate than eGFRcr (13.1%) and eGFRcys (12.0%) but not eGFRcr-cys (8.7%). Combining metabolites, creatinine, cystatin C, and demographics led to the most accurate equation (7.0%). Neither equation had substantial variation among population subgroups. CONCLUSIONS: The new eGFRmet equation could serve as a confirmatory test for GFR estimation.


Assuntos
Cromatografia Líquida/métodos , Taxa de Filtração Glomerular , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Glutamina/análogos & derivados , Glutamina/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Pseudouridina/sangue , Reprodutibilidade dos Testes , Treonina/análogos & derivados , Treonina/sangue , Triptofano/sangue
2.
Artigo em Inglês | MEDLINE | ID: mdl-28029544

RESUMO

Early detection of insulin resistance (IR) and/or impaired glucose tolerance (IGT) is crucial for delaying and preventing the progression toward type 2 diabetes. We recently developed and validated a straightforward metabolite-based test for the assessment of IR and IGT in a single LC-MS/MS method. Plasma samples were diluted with isotopically-labeled internal standards and extracted by simple protein precipitation. The extracts were analyzed by LC-MS/MS for the quantitation of 2-hydroxybutyric acid (0.500-40.0µg/mL), 3-hydroxybutyric acid (1.00-80.0µg/mL), 4-methyl-2-oxopentanoic acid (0.500-20.0µg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50-100µg/mL), oleic acid (10.0-400µg/mL), pantothenic acid (0.0100-0.800µg/mL), and serine (2.50-100µg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1min and the mass spectrometer operated in negative MRM mode. Method validation was performed on three identical LC-MS/MS systems with five runs each. Sufficient linearity (R2>0.99) was observed for all the analytes over the ranges. The imprecision (CVs) was found to be less than 5.5% for intra-run and less than 5.8% for inter-run for the seven analytes. The analytical recovery was determined to be between 96.3 and 103% for the seven analytes. This fast and robust method has subsequently been used for patient sample analysis for the assessment of IR and IGT.

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