Targeted Metabolomics Identifies Reliable and Stable Metabolites in Human Serum and Plasma Samples

Background Information regarding the variability of metabolite levels over time in an individual is required to estimate the reproducibility of metabolite measurements. In intervention studies, it is critical to appropriately judge changes that are elicited by any kind of intervention. The pre-analytic phase (collection, transport and sample processing) is a particularly important component of data quality in multi-center studies. Methods Reliability of metabolites (within-and between-person variance, intraclass correlation coefficient) and stability (shipment simulation at different temperatures, use of gel-barrier collection tubes, freeze-thaw cycles) were analyzed in fasting serum and plasma samples of 22 healthy human subjects using a targeted LC-MS approach. Results Reliability of metabolite measurements was higher in serum compared to plasma samples and was good in most saturated short-and medium-chain acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids and hexose. The majority of metabolites were stable for 24 h on cool packs and at room temperature in non-centrifuged tubes. Plasma and serum metabolite stability showed good coherence. Serum metabolite concentrations were mostly unaffected by tube type and one or two freeze-thaw cycles. Conclusion A single time point measurement is assumed to be sufficient for a targeted metabolomics analysis of most metabolites. For shipment, samples should ideally be separated and frozen immediately after collection, as some amino acids and biogenic amines become unstable within 3 h on cool packs. Serum gel-barrier tubes can be used safely for this process as they have no effect on concentration in most metabolites. Shipment of non-centrifuged samples on cool packs is a cost-efficient alternative for most metabolites.

[1]  M. Schulze,et al.  Variation of serum metabolites related to habitual diet: a targeted metabolomic approach in EPIC-Potsdam , 2013, European Journal of Clinical Nutrition.

[2]  Christian Gieger,et al.  Metabolites associate with kidney function decline and incident chronic kidney disease in the general population. , 2013, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[3]  Andreas Zell,et al.  Preanalytical aspects and sample quality assessment in metabolomics studies of human blood. , 2013, Clinical chemistry.

[4]  J. Adamski,et al.  Targeted Metabolomics of Dried Blood Spot Extracts , 2013, Chromatographia.

[5]  Zeper Abliz,et al.  Liquid chromatography-tandem mass spectrometry-based plasma metabonomics delineate the effect of metabolites' stability on reliability of potential biomarkers. , 2013, Analytical chemistry.

[6]  A. Dirican,et al.  The effect of storage time and freeze-thaw cycles on the stability of serum samples , 2013, Biochemia medica.

[7]  H. M. Draisma,et al.  The Adult Netherlands Twin Register: Twenty-Five Years of Survey and Biological Data Collection , 2013, Twin Research and Human Genetics.

[8]  D. Wishart,et al.  The Metabolomic Profile of Umbilical Cord Blood in Neonatal Hypoxic Ischaemic Encephalopathy , 2012, PloS one.

[9]  D. Wishart,et al.  A metabolomics approach to uncover the effects of grain diets on rumen health in dairy cows. , 2012, Journal of dairy science.

[10]  T. Spector,et al.  Targeted metabolomics profiles are strongly correlated with nutritional patterns in women , 2012, Metabolomics.

[11]  G. Homuth,et al.  A description of large-scale metabolomics studies: increasing value by combining metabolomics with genome-wide SNP genotyping and transcriptional profiling. , 2012, The Journal of endocrinology.

[12]  V. Fellman,et al.  Metabolite Profiles Reveal Energy Failure and Impaired Beta-Oxidation in Liver of Mice with Complex III Deficiency Due to a BCS1L Mutation , 2012, PloS one.

[13]  E. Wolf,et al.  Changing Metabolic Signatures of Amino Acids and Lipids During the Prediabetic Period in a Pig Model With Impaired Incretin Function and Reduced β-Cell Mass , 2012, Diabetes.

[14]  P. Meikle,et al.  Roles of ceramide and sphingolipids in pancreatic β-cell function and dysfunction , 2012, Islets.

[15]  K. Suhre,et al.  Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics , 2012, Metabolomics.

[16]  Christian Baumgartner,et al.  Profiling the human response to physical exercise: a computational strategy for the identification and kinetic analysis of metabolic biomarkers , 2011, Journal of Clinical Bioinformatics.

[17]  Robert Modre-Osprian,et al.  Targeted Metabolomics for Clinical Biomarker Discovery in Multifactorial Diseases , 2011 .

[18]  H. Tanila,et al.  From brain to food: analysis of phosphatidylcholins, lyso-phosphatidylcholins and phosphatidylcholin-plasmalogens derivates in Alzheimer's disease human post mortem brains and mice model via mass spectrometry. , 2011, Journal of chromatography. A.

[19]  Peter Donnelly,et al.  A Genome-Wide Metabolic QTL Analysis in Europeans Implicates Two Loci Shaped by Recent Positive Selection , 2011, PLoS genetics.

[20]  Fabian J Theis,et al.  Discovery of Sexual Dimorphisms in Metabolic and Genetic Biomarkers , 2011, PLoS genetics.

[21]  Florian Kronenberg,et al.  Differences between Human Plasma and Serum Metabolite Profiles , 2011, PloS one.

[22]  T. Pischon,et al.  Reliability of Serum Metabolite Concentrations over a 4-Month Period Using a Targeted Metabolomic Approach , 2011, PloS one.

[23]  Gabi Kastenmüller,et al.  Questionnaire-based self-reported nutrition habits associate with serum metabolism as revealed by quantitative targeted metabolomics , 2011, European Journal of Epidemiology.

[24]  Richard A. Armstrong,et al.  Sample size estimation and statistical power analyses , 2010 .

[25]  T. Hankemeier,et al.  The effect of preanalytical factors on stability of the proteome and selected metabolites in cerebrospinal fluid (CSF). , 2009, Journal of proteome research.

[26]  J. Bruce German,et al.  Effects of sample handling and storage on quantitative lipid analysis in human serum , 2009, Metabolomics.

[27]  David E Bruns,et al.  Stabilization of glucose in blood samples: why it matters. , 2009, Clinical chemistry.

[28]  E. Dennis,et al.  Phospholipase A2 Biochemistry , 2009, Cardiovascular Drugs and Therapy.

[29]  Fabio Garofolo,et al.  Bioanalytical Method Validation , 2004 .

[30]  J. Geleijnse,et al.  High stability of markers of cardiovascular risk in blood samples. , 2003, Clinical chemistry.

[31]  R. Collins,et al.  Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies. , 2003, International journal of epidemiology.

[32]  J Carpenter,et al.  Bootstrap confidence intervals: when, which, what? A practical guide for medical statisticians. , 2000, Statistics in medicine.

[33]  J. Levy,et al.  Within- and between-subject variation in commonly measured anthropometric and biochemical variables. , 1999, Clinical chemistry.

[34]  T. Key,et al.  Stability of vitamins A, C, and E, carotenoids, lipids, and testosterone in whole blood stored at 4 degrees C for 6 and 24 hours before separation of serum and plasma. , 1996, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[35]  A. Folsom,et al.  Short- and long-term repeatability of fatty acid composition of human plasma phospholipids and cholesterol esters. The Atherosclerosis Risk in Communities (ARIC) Study Investigators. , 1995, The American journal of clinical nutrition.

[36]  J. Fleiss,et al.  Intraclass correlations: uses in assessing rater reliability. , 1979, Psychological bulletin.

[37]  J. Ladenson,et al.  Serum versus heparinized plasma for eighteen common chemistry tests: is serum the appropriate specimen? , 1974, American journal of clinical pathology.

[38]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[39]  M. Durán,et al.  Effect of temperature on the stability of long-chain acylcarnitines in human blood prior to plasma separation. , 2007, Clinica chimica acta; international journal of clinical chemistry.

[40]  G. Bonsel,et al.  Can whole-blood samples be stored over 24 hours without compromising stability of C-reactive protein, retinol, ferritin, folic acid, and fatty acids in epidemiologic research? , 2005, Clinical chemistry.

[41]  L. Kaplan,et al.  Clinical Chemistry: Theory, Analysis, and Correlation , 1984 .

[42]  R. K. Cannan,et al.  The creatine-creatinine equilibrium. The apparent dissociation constants of creatine and creatinine. , 1928, The Biochemical journal.

[43]  G. S. Eadie,et al.  THE APPARENT DISSOCIATION CONSTANTS OF CREATINE AND CREATININE , 1926 .