Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules.

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.

Aortic valve replacement with Medtronic Freestyle bioprosthesis: 5-year results.

The Medtronic Freestyle aortic root bioprosthesis has been implanted in patients since August 1992. This study reviews clinical and echocardiographic results at midterm (5 years) after implantation. The Freestyle bioprosthesis was implanted in 1,100 patients in a 21-center Food and Drug Administration clinical trial from August 1992 to October 1998. The device was implanted (1) as a subcoronary valve replacement, (2) as a complete aortic root replacement (full-root), or (3) as a root inclusion. Patients were followed annually by clinical examination and echocardiography. There were 47 deaths early after operation (7.1%). There were 2,478 patient-years of follow-up during which there were 99 deaths or 4.0/pt.-yr. Before implantation, 73% of patients were in New York Heart Association functional class III or IV. After operation, 95% were in class I or II. Transvalvular gradient 4 years after operation was low (7.5+/-5.3 mm Hg) for all valve sizes (subcoronary implant). Small valves (19 and 21 mm) had mean gradients (10.2+/-3.0, 9.1+/-4.4 mm Hg). There was no or mild valve insufficiency in 98% of patients. Actuarial analysis at 5 years showed the rate for freedom from thromboembolism of 86% in subcoronary implant and 93% in full root replacement. Freedom from endocarditis was 98%. Freedom from reoperation for explant of the valve highest in patients having full root replacement (98%) and lower with root inclusion (94%). There were 20 bioprostheses explanted; 10 for endocarditis, 8 for technical reasons, and 2 for structural deterioration. The Medtronic Freestyle bioprosthesis (1) has excellent hemodynamic performance, (2) techniques for insertion that result in a competent valve, (3) low rates of thromboembolism and endocarditis, and (4) a rare rate of structural deterioration at 5 years.

Binding of monovalent anions to human serum transferrin.

Serum transferrin is the protein whose primary function is to bind iron and transport it through the blood. Apotransferrin has two specific metal-binding sites that bind a variety of metal ions in addition to the ferric ion. The distinguishing feature of the transferrins is that a "synergistic" bicarbonate anion is bound along with the metal ion to form a stable Fe(3+)-CO3-Tf ternary complex. Previous research has shown that apotransferrin will also bind divalent anions such as phosphate and sulfate. Difference UV spectroscopy has now been used to show that a series of monovalent anions bind weakly to apotransferrin. Equilibrium constants for the binding of chloride, perchlorate, bromide, fluoride and Hepes have been calculated. A reaction scheme for the binding of anions is proposed which predicts that the binding of the nonsynergistic anions to apotransferrin will interfere with metal binding by competing directly with the binding of the synergistic bicarbonate anion. Difference UV data are presented which demonstrate this type of competition between nonsynergistic anions and Tb3+. Competition from the nonsynergistic anions follows the order HPO4(2-) > SO4(2-) approximately F- > ClO4- approximately Cl- approximately Br-. Speciation calculations have been performed to determine the concentrations of anion-apotransferrin complexes in Hepes and Tris buffers and in human serum and to estimate the extent to which competition from anions in the buffer will interfere with metal-binding to apotransferrin.

Medtronic Freestyle aortic root bioprosthesis: Implant techniques.

BACKGROUND: The Medtronic Freestyle aortic root bioprosthesis is a complete porcine aortic root to allow implantation (1) as a subcoronary valve replacement by removing graft sinus aorta, (2) as a cylinder with the sinotubular junction intact within the aorta (root inclusion), or (3) as a complete aortic root replacement. The choice among the three implant techniques depends on surgeon preference or upon the pathology encountered. The advantages and differences among the three implant techniques are examined. METHODS: The Medtronic Freestyle bioprosthesis was implanted in 1163 patients in a Food and Drug administration (FDA) clinical trial between August 1992 and October 1997. There were 21 centers in the international trial using a single data repository. Clinical data was collected prior to and at operation, at 3 to 6 months and annually. The data were compiled and statistical analysis performed at the data center. RESULTS: Patients having subcoronary valve implants were older (80% > 65 years) and aortic occlusion time was about 20 minutes less than the other methods. Patients having aortic root replacement presented with more aortic valve insufficiency (20%). Pathology of the aortic root and ascending aorta requiring repair was 26%, and larger (27 mm) valves were used in 40% of patients. Risk of operation was lowest (5.0%) with subcoronary valve implants and highest (11.7%) with root replacement technique. Thromboembolism was higher, early and late, with root inclusion (3.0, 3.9%/patient per year) and root replacement (3.2, 3.0%/patient per year) than for subcoronary implants (1.8, 1.6%/patient per year). There were more patients taking warfarin at the 4-year point with root inclusion (20%) or root replacement techniques (24%) than among patients having subcoronary implants (14%). Explants of the valve occurred in 2% of patients, none of whom had aortic root replacement. CONCLUSIONS: The Medtronic Freestyle bioprosthesis is an effective and versatile device for replacement of the aortic valve. It offers implant techniques that can treat the aortic root pathology encountered at surgery and allows the operation to proceed according to surgeon preference.