title: Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin.
abstract: The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.
authors.affiliation: Department of Pharmacology and Toxicology, Consejo Superior de Investigaciones Científicas, Barcelona, Spain.
authors.forename: N
authors.initials: N
authors.lastname: de Vera
authors.forename: R M
authors.initials: RM
authors.lastname: Cristófol
authors.forename: E
authors.initials: E
authors.lastname: Rodríguez Farré
mesh_terms: D000328:Adult; D005978:Glutathione / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008875:Middle Aged; D009638:Norepinephrine / Q000097:blood*; D009961:Orosomucoid / Q000378:metabolism*; D011228:Prealbumin / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D009961:Orosomucoid; D011228:Prealbumin; D012709:Serum Albumin; D006493:Heparin; D005978:Glutathione; D009638:Norepinephrine