title: Impact of high dose vitamin C on platelet function.
abstract: OBJECTIVE
  To examine the effect of high doses of vitamin C (VitC) onex vivohuman platelets (PLTs).
  
  
  METHODS
  Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide. The doses of VitC used here correspond to plasma VitC levels reported in recently completed clinical trials. Prior to supplementation, a baseline sample was collected for analysis. PLTs were sampled again on days 2, 5 and 8 and assayed for changes in PLT function by: Thromboelastography (TEG), for changes in viscoelastic properties; aggregometry, for PLT aggregation and adenosine triphosphate (ATP) secretion in response to collagen or adenosine diphosphate (ADP); and flow cytometry, for changes in expression of CD-31, CD41a, CD62p and CD63. In addition, PLT intracellular VitC content was measured using a fluorimetric assay for ascorbic acid and PLT poor plasma was used for plasma coagulation tests [prothrombin time (PT), partial thrombplastin time (PTT), functional fibrinogen] and Lipidomics analysis (UPLC ESI-MS/MS).
  
  
  RESULTS
  VitC supplementation significantly increased PLTs intracellular ascorbic acid levels from 1.2 mmol/L at baseline to 3.2 mmol/L (Lo VitC) and 15.7 mmol/L (Hi VitC,P< 0.05). VitC supplementation did not significantly change PT and PTT values, or functional fibrinogen levels over the 8 d exposure period (P> 0.05). PLT function assayed by TEG, aggregometry and flow cytometry was not significantly altered by Lo or Hi VitC for up to 5 d. However, PLTs exposed to 3 mmol/L VitC for 8 d demonstrated significantly increased R and K times by TEG and a decrease in the α-angle (P< 0.05). There was also a fall of 20 mm in maximum amplitude associated with the Hi VitC compared to both baseline and day 8 saline controls. Platelet aggregation studies, showed uniform declines in collagen and ADP-induced platelet aggregations over the 8-d study period in all three groups (P> 0.05). Collagen and ADP-induced ATP secretion was also not different between the three groups (P> 0.05). Finally, VitC at the higher dose (3 mmol/L) also induced the release of several eicosanoids including thromboxane B2and prostaglandin E2, as well as products of arachidonic acid metabolismviathe lipoxygenases pathway such as 11-/12-/15-hydroxyicosatetraenoic acid (P< 0.05).
  
  
  CONCLUSIONS
  Alterations in PLT function by exposure to 3 mmol/L VitC for 8 d suggest that caution should be exerted with prolonged use of intravenous high dose VitC.
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Bassem M
authors.initials: BM
authors.lastname: Mohammed
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Kimberly W
authors.initials: KW
authors.lastname: Sanford
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Fisher
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Erika J
authors.initials: EJ
authors.lastname: Martin
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Daniel
authors.initials: D
authors.lastname: Contaifer
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Urszula Osinska
authors.initials: UO
authors.lastname: Warncke
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Dayanjan S
authors.initials: DS
authors.lastname: Wijesinghe
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Chalfant
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Brophy
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Alpha A
authors.initials: AA
authors.lastname: Fowler Iii
authors.affiliation: Bassem M Mohammed, Department of Clinical Pharmacy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Natarajan
keywords: Flow cytometry; Platelet function; Platelet lipidomics; Thromboelastography; Vitamin C