title: The Karlsruhe Metabolomics and Nutrition (KarMeN) Study: Protocol and Methods of a Cross-Sectional Study to Characterize the Metabolome of Healthy Men and Women.
abstract: BACKGROUND
  The human metabolome is influenced by various intrinsic and extrinsic factors. A precondition to identify such biomarkers is the comprehensive understanding of the composition and variability of the metabolome of healthy humans. Sample handling aspects have an important impact on the composition of the metabolome; therefore, it is crucial for any metabolomics study to standardize protocols on sample collection, preanalytical sample handling, storage, and analytics to keep the nonbiological variability as low as possible.
  
  
  OBJECTIVE
  The main objective of the KarMeN study is to analyze the human metabolome in blood and urine by targeted and untargeted metabolite profiling (gas chromatography-mass spectrometry [GC-MS], GC×GC-MS, liquid chromatography-mass spectrometry [LC-MS/MS], and(1)H nuclear magnetic resonance [NMR] spectroscopy) and to determine the impact of sex, age, body composition, diet, and physical activity on metabolite profiles of healthy women and men. Here, we report the outline of the study protocol with special regard to all aspects that should be considered in studies applying metabolomics.
  
  
  METHODS
  Healthy men and women, aged 18 years or older, were recruited. In addition to a number of anthropometric (height, weight, body mass index, waist circumference, body composition), clinical (blood pressure, electrocardiogram, blood and urine clinical chemistry) and functional parameters (lung function, arterial stiffness), resting metabolic rate, physical activity, fitness, and dietary intake were assessed, and 24-hour urine, fasting spot urine, and plasma samples were collected. Standard operating procedures were established for all steps of the study design. Using different analytical techniques (LC-MS, GC×GC-MS,(1)H NMR spectroscopy), metabolite profiles of urine and plasma were determined. Data will be analyzed using univariate and multivariate as well as predictive modeling methods.
  
  
  RESULTS
  The project was funded in 2011 and enrollment was carried out between March 2012 and July 2013. A total of 301 volunteers were eligible to participate in the study. Metabolite profiling of plasma and urine samples has been completed and data analysis is currently underway.
  
  
  CONCLUSIONS
  We established the KarMeN study applying a broad set of clinical and physiological examinations with a high degree of standardization. Our experimental approach of combining scheduled timing of examinations and sampling with the multiplatform approach (GC×GC-MS, GC-MS, LC-MS/MS, and(1)H NMR spectroscopy) will enable us to differentiate between current and long-term effects of diet and physical activity on metabolite profiles, while enabling us at the same time to consider confounders such as age and sex in the KarMeN study.
  
  
  BACKGROUND
  German Clinical Trials Register DRKS00004890; https://drks-neu.uniklinik-freiburg.de/drks_web/navigate.do? navigationId=trial.HTML&TRIAL_ID=DRKS00004890 (Archived by WebCite at http://www.webcitation.org/6iyM8dMtx).
authors.affiliation: Max Rubner-Institut, Department of Physiology and Biochemistry of Nutrition, Karlsruhe, Germany. achim.bub@mri.bund.de.
authors.forename: Achim
authors.identifier: http://orcid.org/0000-0001-6907-5519
authors.initials: A
authors.lastname: Bub
authors.forename: Anita
authors.identifier: http://orcid.org/0000-0002-2398-7245
authors.initials: A
authors.lastname: Kriebel
authors.forename: Claudia
authors.identifier: http://orcid.org/0000-0003-2974-7119
authors.initials: C
authors.lastname: Dörr
authors.forename: Susanne
authors.identifier: http://orcid.org/0000-0001-5150-5904
authors.initials: S
authors.lastname: Bandt
authors.forename: Manuela
authors.identifier: http://orcid.org/0000-0002-7067-2756
authors.initials: M
authors.lastname: Rist
authors.forename: Alexander
authors.identifier: http://orcid.org/0000-0002-5975-0573
authors.initials: A
authors.lastname: Roth
authors.forename: Eva
authors.identifier: http://orcid.org/0000-0003-0554-0025
authors.initials: E
authors.lastname: Hummel
authors.forename: Sabine
authors.identifier: http://orcid.org/0000-0002-9956-2702
authors.initials: S
authors.lastname: Kulling
authors.forename: Ingrid
authors.identifier: http://orcid.org/0000-0003-0698-578X
authors.initials: I
authors.lastname: Hoffmann
authors.forename: Bernhard
authors.identifier: http://orcid.org/0000-0003-0762-7423
authors.initials: B
authors.lastname: Watzl
keywords: cross-sectional studies; healthy subjects; humans; metabolome; metabolomics