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ARS Home » Pacific West Area » Davis, California » Western Human Nutrition Research Center » Obesity and Metabolism Research » Research » Publications at this Location » Publication #355054
Title: Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank

Author
item LA FRANO, MICHAEL - California Polytechnic State University
item CARMICHAEL, SUSAN - Stanford University
item MA, CHEN - Stanford University
item HARDLEY, MACY - Stanford University
item SHEN, TONG - University Of California, Davis
item WONG, RON - Stanford University
item ROSALES, LORENZO - California Polytechnic State University
item BORLOWSKI, KAMIL - University Of California, Davis
item PEDERSEN, THERESA - Advanced Analytics
item SHAW, GARY - Stanford University
item STEVENSON, DAVID - Stanford University
item FIEHN, OLIVER - University Of California, Davis
item Newman, John

Submitted to: Metabolomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2018
Publication Date: 11/15/2018
Citation: La Frano, M.R., Carmichael, S.L., Ma, C., Hardley, M., Shen, T., Wong, R., Rosales, L., Borlowski, K., Pedersen, T.L., Shaw, G., Stevenson, D.K., Fiehn, O., Newman, J.W. 2018. Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank. Metabolomics. 14:151. https://doi.org/10.1007/s11306-018-1450-9.
DOI: https://doi.org/10.1007/s11306-018-1450-9

Interpretive Summary: Population-based biorepositories are important resources for scientific investigations, but sample handling between collection and storage can affect the levels of analytical targets in these samples, and therefore limit what questions can be asked with them. In the present study, we evaluate the impact of delayed sample freezing on the levels of small molecules of biological origins in serum using sample handling protocols that represent a California mid-term pregnancy biobank. Blood collected from non-pregnant healthy female volunteers (n=20) was allowed to clot for 30 min at room temperature and serum was isolated. The serum then underwent three handling protocols: 1) Ideal (IP) - samples frozen (-80 ºC) within 2 hours of collection; 2) Delayed Freezing (DFP) - samples held at room temperature for 3 days and 4ºC for 9 days, the median times for California mid-term pregnancy biobank samples, and then frozen; 3) Delayed Freezing with Freeze-Thaw (DFFTP) – delayed freezing protocol with a freeze-thaw cycle to simulate sub-aliquoting of retrieved samples. Using advanced mass spectrometry techniques, metabolites associated with primary metabolism, a suite of complex lipids, and a series of physiological mediators including oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were measured. The stability of metabolite mean concentrations and their pattern across the three protocols were evaluated, with the ideal protocol as the reference. Sixty-two percent of the 428 identified compounds had good to excellent intraclass correlation coefficients, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, plasmalogen phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze-thaw effects were assay-specific with primary metabolites being primarily impacted. Despite adverse sample handling conditions, numerous small molecules were found to have both stable levels and good concordance, suggesting that while constrained, biorepositories of poorly handled samples are of value.

Technical Abstract: BACKGROUND: Population-based biorepositories are important resources, but sample handling can affect their utility. Here we evaluate the impact of delayed sample freezing on the serum metabolome, using protocols representing a California mid-term pregnancy biobank. METHODS: Blood collected from non-pregnant healthy female volunteers (n=20) underwent three handling protocols after 30 min clotting at room temperature: 1) Ideal (IP) - samples frozen (-80 ºC) within 2 h of collection; 2) Delayed Freezing (DFP) - samples held at room temperature for 3 days and 4ºC for 9 d, the median times for biobank samples, and then frozen; 3) Delayed Freezing with Freeze-Thaw (DFFTP) – delayed freezing protocol with a freeze-thaw cycle to simulate sub-aliquoting of retrieved samples. Mass spectrometry-based metabolomic analyses of primary metabolism and complex lipids along with targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference. RESULTS: Sixty-two percent of the 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze-thaw effects were assay-specific with primary metabolites being primarily impacted. CONCLUSIONS: Despite adverse sample handling conditions, numerous metabolomic compounds had both stable concentrations and good concordance.