title: Heparin-induced release of protein-bound solutes during hemodialysis is an in vitro artifact.
abstract: BACKGROUND
  Several studies have pointed to a release of drugs or protein-bound solutes from their binding sites during heparinization. The effect is attributed to the metabolism of triglycerides to free fatty acids (FFAs), which compete with drugs for protein binding sites. This study evaluated the impact of intradialytic heparin on the free concentration of the uremic toxin p-cresol and on FFAS:
  
  
  METHODS
  Blood samples from hemodialysis (HD) patients, before and during HD, were collected with selected anticoagulation strategies. We assessed the effects of standing time, temperature, pH, and the addition of a lipase inhibitor, tetrahydrolipstatin (THL) to blood samples on the free p-cresol concentration. p-Cresol was analyzed by HPLC with fluorescence detection. We measured FFAs by gas chromatography, and the free fractions of added valproic acid and phenytoin were evaluated by fluorescence polarization immunoassay.
  
  
  RESULTS
  In blood samples (n = 22) not submitted to a specific treatment, free p-cresol increased from 9.9 +/- 5.1 to 31.9 +/- 22.3 micromol/L after 30 min of heparin HD (P < 0.001) and correlated significantly with FFAs (r = 0.80; P = 0.002; n = 12). There was no increase in free p-cresol during heparin-free HD (n = 6) and trisodium citrate HD (n = 9). In addition, p-cresol in ultrafiltrates (n = 3) did not correspond to the free p-cresol in heparinized blood, suggesting that the increase in free p-cresol was artifactual. The release of p-cresol in the test tube was enhanced by standing time (n = 6), sample temperature (n = 6), and alkaline pH (n = 6). Inhibition of lipase activity with THL prevented the increase of FFAs (n = 6) and the release of free p-cresol during HD (n = 22). These results were corroborated by the study of the free fraction of valproic acid (n = 6) and phenytoin (n = 6).
  
  
  CONCLUSIONS
  The free concentrations of protein-bound solutes in plasma of heparinized patients are influenced by external factors that alter the lipase activity in the test tube. The free fraction does not increase during HD when lipase activity is neutralized at the time of blood sampling, so that previously reported increases are probably artifacts.
authors.affiliation: University Hospital Gent, Departments of Internal Medicine, Nephrology Division, and Clinical Biology, B 9000 Gent, Belgium. rita.desmet@rug.ac.be
authors.forename: R
authors.initials: R
authors.lastname: De Smet
authors.forename: J
authors.initials: J
authors.lastname: Van Kaer
authors.forename: H
authors.initials: H
authors.lastname: Liebich
authors.forename: G
authors.initials: G
authors.lastname: Lesaffer
authors.forename: A
authors.initials: A
authors.lastname: Verstraete
authors.forename: A
authors.initials: A
authors.lastname: Dhondt
authors.forename: P
authors.initials: P
authors.lastname: Duym
authors.forename: N
authors.initials: N
authors.lastname: Lameire
authors.forename: R
authors.initials: R
authors.lastname: Vanholder
mesh_terms: D000368:Aged; D000925:Anticoagulants / Q000494:pharmacology* / Q000627:therapeutic use; D016477:Artifacts; D001798:Blood Proteins / Q000378:metabolism*; D001800:Blood Specimen Collection; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D019343:Citric Acid / Q000494:pharmacology; D003408:Cresols / Q000097:blood; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005230:Fatty Acids, Nonesterified / Q000097:blood; D005260:Female; D006403:Hematologic Tests; D006440:Hemofiltration; D006493:Heparin / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D066298:In Vitro Techniques; D007783:Lactones / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors / Q000097:blood; D008297:Male; D008875:Middle Aged; D000077403:Orlistat; D019308:Palmitic Acid / Q000494:pharmacology; D010672:Phenytoin / Q000097:blood; D011485:Protein Binding; D006435:Renal Dialysis; D014635:Valproic Acid / Q000097:blood
chemical_list: D000925:Anticoagulants; D001798:Blood Proteins; D003408:Cresols; D004791:Enzyme Inhibitors; D005230:Fatty Acids, Nonesterified; D007783:Lactones; C032538:4-cresol; D019343:Citric Acid; D019308:Palmitic Acid; D014635:Valproic Acid; D010672:Phenytoin; D006493:Heparin; D000077403:Orlistat; D008049:Lipase