title: Reproducibility and biological variability of HDL's vascular functional assays.
abstract: BACKGROUND
  Recent failures of clinical trials promoting HDL-elevating therapies have prompted research groups to focus on its functional activity in disease. Endothelial effects of HDL can be measured with in vitro cell assays. The reproducibility and biological relevance of these assays have not been explored both in healthy individuals and those at increased cardiovascular (CV) risk.
  
  
  METHODS
  HDL dependent nitric oxide (NO) bioavailability, superoxide (SO) production and serum paraoxonase-1 (PON-1) activity were measured in 35 healthy adults (34.37 24-49) and 8 patients (43.56 37-49) suffering from a chronic inflammatory condition (periodontitis-PD). Assay reproducibility was assessed by independent technicians on consecutive days to determine inter and intra analyser variability for each assay. The 35 healthy individuals were further divided into young (n = 16) and middle aged (n = 19) groups and compared with regards to HDL functions. Within-subject biological variation of HDL function was determined in a sub-group of 25 healthy volunteers at intervals of one day and 1 month, and in 8 patients at intervals of one day and 1 week. Power curves were also generated to estimate the number of patients that would be required for HDL functional assays in a cross-over and parallel study design.
  
  
  RESULTS
  NO bioavailability was the most reproducible assay in healthy adults (coefficient of variation = 1.72%, 1.92 - intra and inter respectively) and PD patients (CV = 4.4% and 5.5%). All measures demonstrated no statistical difference between young and healthy middle aged population. No single assay demonstrated significant variations over time, indicating that within patient variations are negligible. Our power curves for NO bioavailability and PON-1 activity suggest that low number of patients will be required to detect significant differences in HDL function in a cross over and parallel study design.
  
  
  CONCLUSIONS
  This study suggests that in vitro HDL functional assays are reliable and can be used to assess HDL functionality in healthy and diseased populations. NO bioavailability was the most reproducible assay, but PON-1 activity remains the most practical for application in clinical trials due to its capacity for scale.
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Francis
authors.initials: F
authors.lastname: O'Neill
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Eve
authors.initials: E
authors.lastname: McLoughlin
authors.affiliation: Cardiology, Cardiovascular Center, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Meliana
authors.initials: M
authors.lastname: Riwanto
authors.affiliation: Cardiology, Cardiovascular Center, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Manz
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Andreas
authors.initials: A
authors.lastname: Adler
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Sutill
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Tauseef
authors.initials: T
authors.lastname: Khan
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Haywood
authors.affiliation: Institute of Structural & Molecular Biology and London Centre for Nanotechnology, University College London, London, UK.
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.affiliation: Periodontology Unit, Department of Clinical Research, University College London Eastman Dental Institute, London, UK.
authors.forename: Francesco
authors.initials: F
authors.lastname: D'Aiuto
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK.
authors.forename: Marietta
authors.initials: M
authors.lastname: Charakida
authors.affiliation: Cardiology, Cardiovascular Center, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Ulf
authors.initials: U
authors.lastname: Landmesser
authors.affiliation: Vascular Physiology Unit, Institute of Cardiovascular Science, University College London, London, UK; National Institute for Cardiovascular Outcomes Research, University College London, London, UK. Electronic address: j.deanfield@ucl.ac.uk.
authors.forename: John
authors.initials: J
authors.lastname: Deanfield
mesh_terms: D000328:Adult; D000818:Animals; D000886:Anthropometry; D001011:Aorta / Q000473:pathology; D043303:Aryldialkylphosphatase / Q000378:metabolism*; D001774:Blood Chemical Analysis; D002318:Cardiovascular Diseases / Q000097:blood; D002417:Cattle; D002986:Clinical Trials as Topic; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood*; D008297:Male; D009569:Nitric Oxide / Q000737:chemistry*; D010518:Periodontitis / Q000097:blood; D015203:Reproducibility of Results; D013481:Superoxides / Q000097:blood / Q000737:chemistry*; D055815:Young Adult
chemical_list: D008075:Lipoproteins, HDL; D013481:Superoxides; D009569:Nitric Oxide; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human
keywords: High Denisty Lipoprotein; Methodology; Vascular Function