Designing biostable polyurethane elastomers for biomedical implants

The chemical structure, synthesis, morphology, and properties of polyurethane elastomers are briefly discussed. The current understanding of the effect of chemical structure and the associated morphology on the stability of polyurethanes in the biological environments is reviewed. The degradation of conventional polyurethanes appears as surface or deep cracking, stiffening, and deterioration of mechanical properties, such as flex-fatigue resistance. Polyester and poly( tetramethylene oxide) based polyurethanes degrade by hydrolytic and oxidative degradation of ester and ether functional groups, respectively. The recent approaches to develop polyurethanes with improved long-term biostability are based on developing novel polyether, hydrocarbon, polycarbonate, and siloxane macrodiols to replace degradation-prone polyester and polyether macrodiols in polyurethane formulations. The new approaches are discussed with respect to synthesis, properties and biostability based on reported in vivo studies. Among the newly developed materials, siloxane-based polyurethanes have exhibited excellent biostability and are expected to find many applications in biomedical implants.

Long-term in vivo biostability of poly(dimethylsiloxane) / poly(hexamethylene oxide) mixed macrodiol-based polyurethane elastomers

The long-term biostability of a novel thermoplastic polyurethane elastomer (Elast-Eon(TM) 2 80A) synthesized using poly(hexamethylene oxide) (PHMO) and poly(dimethylsiloxane) (PDMS) macrodiols has been studied using an in vivo ovine model. The material's biostability was compared with that of three commercially available control materials, Pellethane(R) 2363-80A, Pellethane(R) 2363-55D and Bionate(R) 55D, after subcutaneous implantation of strained compression moulded flat sheet dumbbells in sheep for periods ranging from 3 to 24 months. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy were used to assess changes in the surface chemical structure and morphology of the materials. Gel permeation chromatography, differential scanning calorimetry and tensile testing were used to examine changes in bulk characteristics of the materials. The results showed that the biostability of the soft flexible PDMS-based test polyurethane was significantly better than the control material of similar softness, Pellethane(R) 80A, and as good as or better than both of the harder commercially available negative control polyurethanes. Pellethane(R) 55D and Bionate(R) 55D. Changes observed in the surface of the Pellethane(R) materials were consistent with oxidation of the aliphatic polyether soft segment and hydrolysis of the urethane bonds joining hard to soft segment with degradation in Pellethane(R) 80A significantly more severe than that observed in Pellethane(R) 55D. Very minor changes were seen on the surfaces of the Elast-Eon(TM) 2 80A and Bionate(R) 55D materials. There was a general trend of molecular weight decreasing with time across all polymers and the molecular weights of all materials decreased at a similar relative rate. The polydispersity ratio, M-w/M-n, increased with time for all materials. Tensile tests indicated that UTS increased in Elast-Eon(TM) 2 80A and Bionate(R) 55D following implantation under strained conditions. However, ultimate strain decreased and elastic modulus increased in the explanted specimens of all three materials when compared with their unimplanted unstrained counterparts. The results indicate that a soft, flexible PDMS-based polyurethane synthesized using 20% PHMO and 80% PDMS macrodiols has excellent long-term biostability compared with commercially available polyurethanes. (C) 2004 Elsevier Ltd. All rights reserved.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Dapsone-induced agranulocytosis. The role of xenobiotic-metabolizing enzymes demonstrated by a case report [Dapson-induzierte agranulozytose. Die rolle fremdstoffmetabolisierender enzyme am beispiel einer kasuistik]

A 34-year-old female patient with a three year history of generalized granuloma annulare was treated systemically with dapsone (DADPS). Six weeks after the onset of treatment, the patient developed an extensive tonsillitis of the base of the tongue with fever and malaise. Routine laboratory work showed a leukocytopenia with agranulocytosis. Further investigation revealed a marked decrease of the enzyme activity of N-acetyltransferase 2, which plays an important role in dapsone metabolism. Treatment included the cessation of dapsone, antibiotic coverage, and G-CSF leading to the rapid improvement of symptoms and normalization of leukocyte counts. Dapsone-induced angina agranulocytotica is a rare event and is interpreted as an idiosyncratic reaction. Depending on genetic polymorphisms of various enzymes, dapsone can be metabolized to immunologically or toxicologically relevant intermediates. Because of the risk of severe hematologic reactions, dapsone should only be employed for solid indications and with appropriate monitoring.

A novel stable inhibitor of endopeptidases EC 3.4.24.15 and 3.4.24.16 potentiates bradykinin-induced hypotension

We have developed a novel inhibitor of the metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16), N-[1-(R, S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), in which alpha-aminoisobutyric acid (Aib) is substituted for an alanine in a well-described but unstable inhibitor, cFP-AAY-pAB. This substitution increases the resistance of the inhibitor to degradation without altering potency. In the present study, we investigated the effects of JA2 (5 mg/kg) on the responses of mean arterial pressure to bradykinin, angiotensin I, and angiotensin II in conscious rabbits. The depressor responses to both low (10 ng/kg) and high (100 ng/kg) doses of bradykinin were increased 7.0 +/- 2.7-fold and 1.5 +/- 0.3-fold, respectively, during the 30 minutes after JA2 administration (mean+/-SEM, n=8). Bradykinin potentiation was undiminished 4 hours after JA2 injection. In contrast, the hypertensive effects of angiotensins I and II were unaltered, indicating that the bradykinin-potentiating effects were not due to angiotensin-converting enzyme inhibition. These data suggest that JA2 is not only a potent and specific inhibitor of EP24.15 and EP24.16 but is also stable in vivo. Furthermore, the potentiation of bradykinin-induced hypotension by JA2 suggests for the first time a role for one or both of these peptidases in the metabolism of bradykinin in the circulation.

Emotional distress induced rhabdomyolysis in an individual with carnitine palmitolytransferase deficiency

A 48-year-old male patient with underlying CPT II enzyme deficiency is described. Emotional stress appeared to precipitate recurrent myalgias, rhabdomyolysis and reversible renal impairment over a 40-year period. Our search of the English literature indicates this to be the first time that the emotional stress has been documented to precipitate the CPT II syndrome. Although the pathogenesis of this syndrome has yet to be established, existing knowledge is briefly reviewed and the likely metabolic and neuroendocrine mechanisms which link emotional stress to muscle metabolism are examined. These mechanisms influence the extent of lipolysis or glycolysis that occurs during the process of muscle ATP generation. It is suggested that neuroendocrine and other stress related changes which favour lipolysis over glycolysis adversely effect muscle energy metabolism in patients whose mitochondria are deficient in CPT II enzyme. Possible treatment strategies are those that favour glycolysis over fatty acid metabolism and include a variety of ways of modulating sympathetic and parasympathetic tone. The use of carbohydrate supplementation P-blockers and anxiolytic agents is discussed.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products. (C) 2003 Elsevier Science B.V. All rights reserved.

Evidence for induced microsomal bilirubin degradation by cytochrome P450 2A5

Oxidative metabolism of bilirubin (BR) - a breakdown product of haem with cytoprotective and toxic properties - is an important route of detoxification in addition to glucuronidation. The major enzyme(s) involved in this oxidative degradation are not known. In this paper, we present evidence for a major role of the hepatic cytochrome P450 2A5 (Cyp2a5) in BR degradation during cadmium intoxication, where the BR levels are elevated following induction of haem oxygenase-1 (HO-1). Treatment of DBA/2J mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was substantial at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of HO-1 preceded that of Cyp2a5 with a 5-10 h interval. BR totally inhibited the microsomal Cyp2a5-dependent coumarin hydroxylase activity, with an IC50 approximately equal to the substrate concentration. The 7-methoxyresorufin 7-O-demethylase (MROD) activity, catalyzed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and a monoclonal antibody against the Cyp2a5 by about 90%. Furthermore, 7-methoxyresorufin, a substrate for the Cyp1a2, inhibited BR degradation activity by approximately 20%. In sum, the results strongly suggest a major role for Cyp2a5 in the oxidative degradation of BR. Secondly, the coordinated up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a network of enzyme systems in the maintenance of balancing BR production and elimination.

Thiamin nutrition and catalysis-induced instability of thiamin diphosphate

Thiamin (vitamin B1) is required in animal diets because it is the precursor of the enzyme cofactor, thiamin diphosphate. Unlike other B vitamins, the dietary thiamin requirement is proportional to non-fat energy intake but there is no obvious biochemical reason for this relationship. In the present communication we show for two enzymes that the cofactor undergoes a slow destruction during catalysis, which may explain the interdependence of thiamin and energy intakes.

The sites of neural adaptation induced by resistance training in humans

Although it has long been supposed that resistance training causes adaptive changes in the CNS, the sites and nature of these adaptations have not previously been identified. In order to determine whether the neural adaptations to resistance training occur to a greater extent at cortical or subcortical sites in the CNS, we compared the effects of resistance training on the electromyographic (EMG) responses to transcranial magnetic (TMS) and electrical (TES) stimulation. Motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of 16 individuals before and after 4 weeks of resistance training for the index finger abductors (n = 8), or training involving finger abduction-adduction without external resistance (n = 8). TMS was delivered at rest at intensities from 5 % below the passive threshold to the maximal output of the stimulator. TMS and TES were also delivered at the active threshold intensity while the participants exerted torques ranging from 5 to 60 % of their maximum voluntary contraction (MVC) torque. The average latency of MEPs elicited by TES was significantly shorter than that of TMS MEPs (TES latency = 21.5 ± 1.4 ms; TMS latency = 23.4 ± 1.4 ms; P < 0.05), which indicates that the site of activation differed between the two forms of stimulation. Training resulted in a significant increase in MVC torque for the resistance-training group, but not the control group. There were no statistically significant changes in the corticospinal properties measured at rest for either group. For the active trials involving both TMS and TES, however, the slope of the relationship between MEP size and the torque exerted was significantly lower after training for the resistance-training group (P < 0.05). Thus, for a specific level of muscle activity, the magnitude of the EMG responses to both forms of transcranial stimulation were smaller following resistance training. These results suggest that resistance training changes the functional properties of spinal cord circuitry in humans, but does not substantially affect the organisation of the motor cortex.

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Enzyme immobilization in porous solid supports - penetration of immobilized enzyme

The process of enzyme immobilization under the diffusion-controlled regime (i.e., fast attachment of enzyme compared to its diffusion) is modeled and theoretically solved in this article. Simple and compact solutions for the penetration depth of immobilized enzyme and the bulk enzyme concentration versus time are presented. Furthermore, the conditions for the validity of our solutions are also given in this article so that researchers can discover when the theoretical solutions can be applied to their systems.

Effect of pH on Na induced Ca deficiency

Although it is well known that high Na concentrations induce Ca deficiency in acidic conditions, the effect of high pH on this competitive mechanism is not so well understood. The effect of Ca activity ratio (CAR) and pH on the Ca uptake of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald) and Rhodes grass (Chloris gayana cv. Pioneer) in Na dominated solution cultures and in soil was investigated. Changes in pH in the alkaline range were shown not to affect the critical CAR of 0.024 (corresponding to 90 % relative root length) for mungbeans grown in solution culture. Results from soil grown mungbeans confirmed those from solution culture, with a critical CAR of 0.025. A critical CAR of 0.034 was also established for soil grown Rhodes grass. The similarity of critical values established for mungbeans and Rhodes grass in solution culture and soil justifies the use of both solution culture and soil solution measurement as techniques for studying plant growth and limitations across plant species.

Mg induced Ca deficiency under alkaline conditions

Little is known about Mg induced Ca deficiency in alkaline conditions, and the relationship between Mg induced Ca deficiency and Na induced Ca deficiency. Dilute nutrient solutions (dominated by Mg) were used to investigate the effect of Ca activity ratio (CAR) on the growth of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald). At pH 9.0, root growth was reduced below a critical CAR of 0.050 (corresponding to 90 % relative root length). Root growth was found to be limited more in Mg solutions than had been previously observed for Na solutions. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for both Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present.