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"PROJECT":{"PROJECT_TITLE":"Improving kidney function assessment in health and disease","PROJECT_SUMMARY":"Use results from a global metabolomics platform with existing measured GFR assessments to identify a set of markers from which to develop a highly accurate targeted mass spectrometry multiplex assay","INSTITUTE":"Tufts Medical Center","DEPARTMENT":"Nephrology","LABORATORY":"Metabolon; University of Minnesota","LAST_NAME":"Inker","FIRST_NAME":"Lesley","ADDRESS":"800 Washington Street, Boston, Massachusetts, 02111, USA","EMAIL":"Lesley.Inker@tuftsmedicine.org","PHONE":"6176368783","FUNDING_SOURCE":"NIDDK"},

"STUDY":{"STUDY_TITLE":"Evaluation of Novel Candidate Filtration Markers from a Global Metabolomics Discovery for Glomerular Filtration Rate Estimation (ALTOLD)","STUDY_SUMMARY":"Background: Creatinine and cystatin-C are recommended for estimating glomerular filtration rate (eGFR) but accuracy is suboptimal. Using untargeted metabolomics data, we sought to identify candidate filtration markers using a novel approach based on their maximal joint association with measured GFR (mGFR) with flexibility to consider their biological and chemical properties later. Methods: We analyzed metabolites measured in seven diverse studies of 2,851 participants on the Metabolon H4 platform that had Pearson correlations with log mGFR <-0.5. We used a stepwise approach to develop models to estimate mGFR including two to 15 metabolites with and without inclusion of creatinine and demographics. We then selected candidate filtration markers from those metabolites found >20% in models that did not demonstrate substantial overfitting in cross-validation and with small (<0.1 in absolute value) coefficients for demographics. Results: In total, 456 named metabolites were present in all studies, and 36 had correlations <-0.5 with mGFR. We developed 2,225 models including these metabolites; all had lower RMSEs and smaller coefficients for demographic variables compared to estimates using untargeted creatinine. Cross-validated RMSEs (0.187-0.213) were similar to original RMSEs for models with ≤ 10 metabolites. Our criteria identified 17 metabolites, including 12 new candidate filtration markers. Conclusion: We identified candidate metabolites with maximal joint association with mGFR and minimal association with demographic variables across varied clinical settings. Future analyses will assess metabolite biological and chemical characteristics in the path towards development of a panel eGFR that is more accurate and less reliant on demographic variables than current eGFR. ACRONYMS AASKG1: African American Study of Kidney (patient data at G1 visit). ALTOLD: Assessing Long Term Outcomes in Living Kidney Donors study. MDRD: The Modification of Diet in Renal Disease study.","INSTITUTE":"Tufts Medical Center","DEPARTMENT":"Nephrology","LAST_NAME":"Inker","FIRST_NAME":"Lesley","ADDRESS":"800 Washington Street","EMAIL":"Lesley.Inker@tuftsmedicine.org","PHONE":"6176368783"},

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{
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"Subject ID":"-",
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{
"Subject ID":"-",
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"Subject ID":"-",
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{
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{
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"Subject ID":"-",
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"Subject ID":"-",
"Sample ID":"6568784",
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{
"Subject ID":"-",
"Sample ID":"6568785",
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{
"Subject ID":"-",
"Sample ID":"6568786",
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},
{
"Subject ID":"-",
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{
"Subject ID":"-",
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{
"Subject ID":"-",
"Sample ID":"6568789",
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"Subject ID":"-",
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],
"COLLECTION":{"COLLECTION_SUMMARY":"samples were obtained from NIDDK repository","SAMPLE_TYPE":"Blood (plasma)"},

"TREATMENT":{"TREATMENT_SUMMARY":"Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files are archived and extracted as described below"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"High pH (LC/MS Neg)","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)","SOLVENT_A":"-","SOLVENT_B":"-","FLOW_GRADIENT":"-","FLOW_RATE":"-","COLUMN_TEMPERATURE":"-"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"Other","ION_MODE":"NEGATIVE","MS_COMMENTS":"Metabolon (LC/MS Neg)"},

"MS_METABOLITE_DATA":{
"Units":"raw area counts rescaled to set the median equal to 1",

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}

}