title: HPLC fractions of human uremic plasma inhibit the RBC membrane calcium pump.
abstract: We have reported that uremic plasma filtrates (UF) inhibit the red blood cell (RBC) membrane calcium pump. The inhibitor was dialyzable, smaller than 3,000 molecular weight, heat-stable, and protease-resistant. In the present study, we used reverse-phase preparative HPLC, analytical HPLC, and Sephadex G-25 elution to identify inhibitory fractions. Inhibition was confirmed in three different bioassays: (1) Sr2+ efflux in intact RBC, the primary bio-assay; (2) 45Ca efflux in intact RBC; and (3) calcium ATPase activity in isolated RBC membranes. Active fractions were analyzed by mass spectrometry, capillary electrophoresis, enzymatic analysis, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy. These demonstrated a number of compounds, including: sugars, polyols, osmolytes like betaine and myoinositol, amino acids, and other metabolites, such as 3-D-hydroxybutyrate, dimethylglycine, trimethylamine-N-oxide, guanidinoacetic acid and glycine. Many individual compounds were then tested for an effect on the calcium pump. Thus, HPLC was able to separate a substantial number of compounds in inhibitory fractions. Efforts are under way for precise identification of the inhibitor, to advance our understanding of uremic toxicity and/or hypertension in CRF.
authors.affiliation: Department of Medicine, Veterans Affairs Medical Center and University of Washington School of Medicine, Seattle 98108, USA.
authors.forename: A
authors.initials: A
authors.lastname: Lindner
authors.forename: R
authors.initials: R
authors.lastname: Vanholder
authors.forename: R
authors.initials: R
authors.lastname: De Smet
authors.forename: T R
authors.initials: TR
authors.lastname: Hinds
authors.forename: P
authors.initials: P
authors.lastname: Vogeleere
authors.forename: P
authors.initials: P
authors.lastname: Sandra
authors.forename: P
authors.initials: P
authors.lastname: Foxall
authors.forename: S
authors.initials: S
authors.lastname: Ringoir
mesh_terms: D000328:Adult; D000368:Aged; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D008875:Middle Aged; D013324:Strontium / Q000097:blood; D014511:Uremia / Q000097:blood*
chemical_list: D004791:Enzyme Inhibitors; D000252:Calcium-Transporting ATPases; D002118:Calcium; D013324:Strontium