Robert Zand

Prevention of bioprosthetic heart valve calcification by ethanol preincubation: Efficacy and mechanisms

BACKGROUND Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. METHODS AND RESULTS Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. CONCLUSIONS Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Mechanisms of bioprosthetic heart valve failure: Fatigue causes collagen denaturation and glycosaminoglycan loss

Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.

Circular dichroism studies: I. Cytochrome c

Abstract The circular dichroism spectra of the ferri and ferro forms of cytochrome c obtained from horse, beef, chicken, turtle, and Pseudomonas aeruginosa have been measured at pH 7.0 over the wavelength region from 690 to 220 mμ. For purposes of analysis the spectra were arbitrarily divided into five regions: region I, 690-580 mμ; region II, 580-450 mμ (region related to heme coordination); region III, 450-350 mμ (Soret region); region IV, 350-240 mμ (region of protein-heme interactions and Cotton effects due to the aromatic side-chain residues); and region V, below 240 mμ (region of peptide bond absorption). The spectra are complex, exhibiting numerous Cotton effects. The molar ellipticities of all five cytochromes are small and tend to increase in magnitude at shorter wavelengths. The circular dichroism spectra of the mammalian-type cytochrome c of horse, beef, chicken, and turtle are grossly similar to each other. The similarities and differences between the spectra of the mammalian-type cytochromes c were utilized to locate the positions of some of the optically active transitions in regions II, III, and IV. The positions of the two Cotton effects at 280–290 mμ associated with the aromatic side-chain residues of the protein appear to be unaffected by change in oxidation. The spectra of all five ferrocytochromes c are alike in regions II and III. The spectra of the ferri forms of the mammalian-type and Pseudomonas cytochrome c in the Soret region appear to be mirror images of each other. This effect must reflect a difference in the environment of the heme group in the ferricytochromes c of Pseudomonas and mammalian type. Since the effect does not occur in the spectra of the ferrocytochromes c examined, it is likely that the environment of the heme group in the reduced form of cytochrome c remains invariant. An analysis of the relationship of the circular dichroism of all five ferri- and ferrocytochromes c in regions II and III to the structure of the central coordination complex of mammalian-type cytochrome c is presented.

Prevention of calcification of glutaraldehyde-crosslinked porcine aortic cusps by ethanol preincubation: mechanistic studies of protein structure and water-biomaterial relationships.

Clinical usage of bioprosthetic heart valves (BPHVs) fabricated from glutaraldehyde-pretreated porcine aortic valves is restricted due to calcification-related failure. We previously reported a highly efficacious ethanol pretreatment of BPHVs for the prevention of cuspal calcification. The aim of the present study is to extend our understanding of the material changes brought about by ethanol and the relationship of these material effects to the ethanol pretreatment anticalcification mechanism. Glutaraldehyde-crosslinked porcine aortic valve cusps (control and ethanol-pretreated) were studied for the effects of ethanol on tissue water content and for spin-lattice relaxation times (T1) using solid state proton NMR. Cusp samples were studied for protein conformational changes due to ethanol by ATR-FTIR spectroscopy. The changes in cuspal tissue-cholesterol (in vitro) interactions also were studied. Cusp material stability was assessed in terms of residual glutaraldehyde content and collagenase degradation. Water content of the cusp samples was decreased significantly due to ethanol pretreatment. The cuspal collagen conformational changes (per infrared spectroscopy) brought about by ethanol pretreatment were persistent even after rat subdermal implantation of cusp samples for 7 days. In vitro cholesterol uptake by cusps was greatly reduced as a result of ethanol pretreatment. Ethanol pretreatment of cusps also resulted in increased resistance to collagenase digestion. Cuspal glutaraldehyde content was not changed by ethanol pretreatment. We conclude that ethanol pretreatment of bioprosthetic heart valve cusps causes multi-component effects on the tissue/material and macromolecular characteristics, which partly may explain the ethanol-pretreatment anticalcification mechanism.

Inhibition of aortic wall calcification in bioprosthetic heart valves by ethanol pretreatment: Biochemical and biophysical mechanisms

The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80+/-8.45 microg/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90+/-7.24 microg/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02+/-4.42 microg/mg compared to the control calcium level of 56.35+/-6.14 microg/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T1) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.

Bilirubin binding to myelin basic protein, histones and its inhibition in vitro of cerebellar protein synthesis

Summary The binding of unconjugated bilirubin to bovine CNS myelin basic protein and to lysine and arginine rich histones has been demonstrated by means of difference spectroscopy and circular dichroism spectroscopy. This is the first demonstration of a brain specific protein that can bind bilirubin and provides a mechanism for bilirubin retention in brain as well as a mechanism for interfering with the normal acidic lipid-basic protein binding interaction. The inhibition of protein synthesis in cerebellar homogenates by bilirubin has also been demonstrated. The inhibition is about 50 per cent in the presence of 100 μM bilirubin and 85 per cent in the presence of 700 μM bilirubin, These results require that the current mechanisms for bilirubin neurotoxicity and cytotoxicity be expanded to include the present findings.

α‐AMINOCYCLIC AND BICYCLIC ALKANE CARBOXYLIC ACIDS: DIFFERENTIAL EFFECTS ON SELECTED AMINO ACIDS OF RAT BRAIN CORTEX1

Abstract— The intraperitoneal administration of 1‐aminocyclopentane carboxylic acid, 1‐aminocyclohex‐ane carboxylic acid, l‐aminocycloheptane carboxylic acid, 1‐aminocyclooctane carboxylic acid, exo‐2‐aminobicyclo(2,2. l)heptane‐2‐carboxylic acid. endo‐2‐aminobicyclo(2,2.1)heptane‐2‐carboxylic acid. 2‐aminobicyclo(2.2.2)octane‐2‐carboxylic acid and 2‐aminobicyclo(3,2.l)octane‐2‐carboxylic acid to 18‐day‐old male rats selectively perturbed the levels of neutral amino acids in the cerebral cortex. While the effect of the above compounds was rather diversified and usually resulted in a reduction of amino acid levels. marked elevations of the levels of valine and isoleucine were also noted. 1‐Aminocycloheptane and cyclooctane carboxylic acids were particularly noteworthy, in that they elicited a marked reduction of the levels of cortical phenylalanine.

Studies of posttranslational modifications in spiny dogfish myelin basic protein

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.

Synthesis and characterization of thermally stable polymers containing phenazine

The synthesis and characterization of two thermally stable polymers containing a polybenzimidazole and a ladder polymer structure are described. The tetraamino derivative of phenazine required for these syntheses was obtained by the reduction of the commercially available diamino, dinitro derivative of benzene. The polybenzimidazole polymer was synthesized by condensing tetraaminophenzine with terephthaldehyde in dimethylacetamide solvent. The resulting polymer had an intrinsic viscosity of 0.94 g/dl in methanesulfonic acid solvent. The TGA of the polymer showed it to be stable up to 4708C. The ladder polymer was prepared by reacting tetramainophenazine with dihydroquinone in polyphosphoric acid at a temperature of 1468C. The polymer formed a brittle, free-hanging film when cast from methanesulfonic acid. The molecule exhibited high degree of crystallinity and had a tendency to form dendritic-shaped structures. Doping with 15% iodine resulted in a conductivity of 9 £ 10 27 mho/cm. It was thermally stable in air, showing a 10% weight loss at 4108C. q 1999 Elsevier Science Ltd. All rights reserved.

Circular dichroism of ferricytochrome c

Abstract The circular dichroism (CD) spectra (650 − 220 mu) of horse heart ferricytochrome c, Pseudomonas aeruginosa ferricytochrome c and ferriheme octapeptide from horse heart cytochrome c have been measured at pH 7. The CD spectra were compared to the optical rotatory dispersion (ORD) spectra and it was found that some of the optically active transitions responsible for the multiple extrinsic Cotton effect observed previously in the ORD spectra of cytochrome c, could be resolved. Of special interest was the observation that the band shapes of the two proteins in the 480 − 410 mu region, appear to be approximate mirror images of one another. This phenomenon is most likely an indication of the arrangement of ligands in positions 5 and 6 of the heme group.