title: High-throughput 1H NMR-based metabolic analysis of human serum and urine for large-scale epidemiological studies: validation study.
abstract: BACKGROUND
  Metabolic profiling of biofluid specimens is an established method for investigating disease states in clinical studies but is only recently being applied to large-scale human population studies. As part of protocol development for the UK Biobank study, a (1)H nuclear magnetic resonance (NMR)-based metabonomic analysis of specimen storage effects and analytical reproducibility was carried out using urine and serum specimens from 40 volunteers.
  
  
  METHODS
  Aliquots of each specimen were stored for t = 0 and t = 24 h at 4 degrees C prior to freezing, and in the case of serum samples for a further 12 h (t = 36), to determine whether the storage times affected specimen composition and quality. A blinded split-specimen matching exercise was implemented to assign candidate spectral pairs stored for different times using multivariate statistical analysis of the NMR data.
  
  
  RESULTS
  Using a chemometric strategy, split specimens at time t = 0 and t = 24 or 36 h after storage at 4 degrees C were easily paired and the split-specimen matching task was reduced to a workable size. (1)H NMR profiling established that the t = 24 h urine and serum groups showed no systematic metabolite changes, indicating biochemical stability. Some small differences in serum specimens stored for t = 36 h at 4 degrees C were detectable only by multivariate analysis, and were attributed to generalized alterations in proteins and protein fragments, and possibly trimethylamine-N-oxide. No other specific metabolite was implicated.
  
  
  CONCLUSIONS
  For the purposes of NMR-based analysis, storage of urine and serum for up to t = 24 h at 4 degrees C does not detectably affect the metabolic profile and the methodology is robust. Future application of multivariate methods to data-rich studies should substantially enhance information recovery from epidemiological studies.
authors.affiliation: Biomolecular Medicine, SORA Division, Faculty of Medicine, Imperial College London, Exhibition Road, London SW7 2AZ, UK. r.barton@ic.ac.uk
authors.forename: Richard H
authors.initials: RH
authors.lastname: Barton
authors.forename: Jeremy K
authors.initials: JK
authors.lastname: Nicholson
authors.forename: Paul
authors.initials: P
authors.lastname: Elliott
authors.forename: Elaine
authors.initials: E
authors.lastname: Holmes
mesh_terms: D018070:Biological Specimen Banks; D001774:Blood Chemical Analysis / Q000592:standards*; D015925:Cryopreservation; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010865:Pilot Projects; D017410:Practice Guidelines as Topic; D044967:Serum / Q000737:chemistry*; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D006113:United Kingdom; D016482:Urinalysis / Q000592:standards*; D014556:Urine / Q000737:chemistry*