Complications of periacetabular osteotomy.

There was a statistically significant decrease in major complications from 17% to 2.9% when comparing the first 35 cases with the second 35 cases of periacetabular osteotomy performed by one surgeon. There were no cases of intraarticular fracture, conversion to total hip replacement, or deaths in this series. Of considerable significance was that almost all major complications, as defined for disclosure in this report, left the patients with no permanent sequelae after either successful treatment, as in intraoperative bleeding, or with observation with time, as for recovery of sciatic nerve function. The complication rate of periacetabular osteotomy decreases significantly in proportion to increasing experience, as documented in this study. Patients in ongoing studies completed the Western Ontario and McMaster Universities Osteoarthritis Index and the Short Form- 36 preoperatively, which will add to the authors' ability to comment on functional outcomes in future reports.

Functional recovery following neurorrhaphy of the rat sciatic nerve by epineurial repair compared with tubulization.

Recovery of motor function is often poor following transection injuries to peripheral nerves. The purpose of this study was to measure and compare functional recovery of the sciatic nerve in the rat following transection and neurorrhaphy with the use of a nerve guide tube and with traditional end-to-end epineurial repair. Muscle recovery was also evaluated following a crush injury, a model of an axonotmetic lesion. Recovery was assessed at 8, 16, and 32 weeks after injury by measuring the isometric contractile properties of the soleus muscle and at 8 and 16 weeks by measuring the conduction properties of the sciatic nerve. The mean conduction velocity of the sciatic nerve in the crush group and both transection groups was significantly slower than that of controls at both 8 and 16 weeks. Following a transection injury, the soleus became a significantly faster muscle as measured by time to peak twitch tension. By 32 weeks, the maximum isometric tension of the soleus muscle recovered to 90% that of the control group following a crush injury and to less than 70% following a transection injury and repair. Recovery was better in the epineurial repair group than in the tube repair group at 8 weeks, but no difference was found between the groups at 16 or 32 weeks. These results demonstrate that nerve guide tubes are a potential alternative to epineurial repair. The poor motor recovery following repair of transection injuries may be related to poor specificity of reinnervation.

Calcium measurement in isolated arterioles during myogenic and agonist stimulation.

Vascular smooth muscle calcium was measured during agonist treatment or pressure-induced stimulation of the myogenic response in isolated first-order skeletal muscle arterioles. Arterioles (40-180 microns) with spontaneous tone were isolated from rat cremaster muscle and cannulated. Arterioles were loaded with the calcium-sensitive dye fura-2 and excited at 340 and 380 nm. Images of vessel fluorescence were formed with a fluorescence microscope and digitized using an image processor coupled to a low light level camera. The fluorescent images allowed individual vascular smooth muscle cells to be seen within the arteriolar wall. Fluorescent intensity of the vessel wall, expressed as the ratio of fluorescence at 340 nm/380 nm, was used to estimate changes in vessel wall calcium. Topical application of norepinephrine (10 microM) to the arterioles caused a rapid and sustained constriction of the arterioles (64% of basal diam). The calcium response was biphasic consisting of a transient spike to 271% of basal followed by a decrease to a new steady state at 143% of basal. In comparison, steady-state indolactam (1 microM) produced a similar degree of constriction without an increase in calcium. Adenosine significantly dilated (35%) the arterioles and produced a decrease (24%) in vessel wall calcium. To investigate the myogenic response, intravascular pressure was step increased from 90 to 130 cmH2O. Increasing intravascular pressure caused an initial increase in vessel diameter of approximately 5% followed by active constriction that returned diameter to basal diameter. In association with this diameter change, estimated vessel wall calcium increased rapidly 8 +/- 2% and then continued to increase more slowly and remained elevated at 10-15% above basal levels. This study demonstrates the successful application of calcium-imaging technology in isolated arterioles for study of the role of calcium in arteriolar function. Results indicate that the calcium-contraction relationship differs for different agonists and are further consistent with a role for pressure-induced increases in vascular smooth muscle calcium during the myogenic response.

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

The inhibition of the membrane-bound enzyme cytochrome c oxidase by aliphatic n-alcohols and other neutral organic compounds was studied as a model for anaesthetic action and drug toxicity. The n-alcohols (C1 to C14) displayed a variation in inhibition constant of over 500,000-fold. The inhibition constants correlated well with the number of carbon atoms in the n-alcohols and also their n-octanol/water partition coefficients. General anaesthetic potency is known to be similarly well correlated with octanol/water partition coefficients. The free-energy change for transferring a methylene group of the n-alcohol to the more hydrophobic environment bound to the enzyme is similar to that for transferring a methylene group from water to pure alcohol. These results are consistent with the n-alcohols inhibiting by binding to an octanol-like environment on the enzyme or the protein/phospholipid interface. Neither negatively charged carboxylates nor positively charged amine analogues were observed to cause any inhibition, indicating that this postulated binding site may be uncharged. Inhibition of cytochrome c oxidase by n-alcohols was also demonstrated in both bovine heart and rat liver sonicated submitochondrial fragments.

The Adriamycin (doxorubicin)-induced inactivation of cytochrome c oxidase depends on the presence of iron or copper.

1. It was confirmed that Adriamycin (doxorubicin) inactivates cytochrome c oxidase upon incubation. However, further investigation shows that this inactivation is strongly dependent upon the presence of Fe3+ and Cu2+. Trace amounts of these transition metal ions, present in phosphate and Tris buffers, bind strongly to the Adriamycin and the complex formed is responsible for the inactivation of cytochrome c oxidase. No Adriamycin-induced inactivation of cytochrome c oxidase occurred in the presence of EDTA or in phosphate buffers purified on a cation exchange column to remove trace metals. 2. The metal ion-induced inactivation of cytochrome c oxidase by Adriamycin results in significant decreases in both the maximum velocity and the Michaelis constant. The degree of inactivation is strongly dependent on the Fe3+ concentration. 3. Cardiolipin partially protects against cytochrome c oxidase inactivation, presumably by binding to the cytochrome c oxidase, whereas catalase or superoxide dismutase partially protect by scavenging damaging reactive oxygen species generated within a Fe3+-Adriamycin-enzyme complex.

Adriamycin and its iron(III) and copper(II) complexes. Glutathione-induced dissociation; cytochrome c oxidase inactivation and protection; binding to cardiolipin.

Some reactions of adriamycin (doxorubicin) and its Fe3+ and Cu2+ complexes were investigated with a view to understanding the mechanisms by which metal ion-adriamycin complexes damage cellular components. The ability of adriamycin in the presence of Cu2+ to inactivate the mitochondrial enzyme cytochrome c oxidase was effectively prevented by physiologic levels of glutathione. This result is explained by the observation that glutathione reacts with the Cu2+-adriamycin complex to produce free adriamycin. As sulfhydryl compounds are, in contrast, known to promote Fe3+-adriamycin-induced damage to cellular components, these results suggest that the response of a metal ion-adriamycin system to the presence of sulfhydryl compounds may be indicative of whether or not Cu2+-adriamycin is the damaging species. The partition of adriamycin into the octanol phase of an octanol-water two-phase system was greatly enhanced by the presence of cardiolipin. This result can be explained by the formation of a strong adriamycin-cardiolipin complex in the octanol phase which is one-half formed at an adriamycin concentration of 6 microM.

The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation.

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.

The kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase in solvents of increased viscosity are partially diffusion controlled.

The steady-state spectrophotometrically determined initial velocity kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase were examined for effects of diffusion control in solvents of increased viscosity. Both glycerol/water and sucrose/water proved unsatisfactory as viscosogens due to weak competitive inhibition (Ki values of 2.6 M and 1.6 M, respectively). However, polyethylene glycol (PEG) was satisfactory as a viscosogen. The measured diffusion coefficient of ferrocytochrome c in PEG/water was shown to follow closely the Stokes-Einstein equation. In PEG/water mixtures at high ionic strength the minimum association rate constant (kmin = Vmax/(Km[EO]) is partly diffusion controlled with contributions from diffusion control and chemical activation control being about equal at 5 mPa X s, a viscosity that may be physiologically relevant. This finding can be interpreted to mean that cytochrome c oxidase is an enzyme that has evolved to approach its maximum efficiency. The steady-state kinetics were also examined at low ionic strength where multiphasic kinetics are exhibited. The effect of increased viscosity was exhibited over the whole experimentally accessible region indicating that there are effects due to diffusion control on both the high-affinity and low-affinity binding of ferrocytochrome c. Several models for diffusion control were examined and a comparison is made with other diffusion-controlled reactions of proteins.

The association reaction of yeast alcohol dehydrogenase with coenzyme is partly diffusion-controlled in solvents of increased viscosity.

The steady-state kinetics of the yeast and liver alcohol dehydrogenase catalyzed reduction of aldehydes were examined in solvent mixtures of increased viscosity. This was done to investigate the effects of diffusion control on the fast association of NADH with the enzymes. Both glycerol and sucrose were unsatisfactory as viscosogens, as they inhibited the enzyme, but poly(ethylene glycol)/water mixtures were satisfactory. The 5-fold faster reaction of yeast alcohol dehydrogenase with NADH is partly diffusion controlled, whereas the slower liver alcohol dehydrogenase reaction showed no diffusion effects. These results are consistent with a yeast alcohol dehydrogenase active site that has relatively little steric hindrance to NADH binding. It is estimated that contributions to this association reaction from diffusion control and chemical activation control are equal at a solvent viscosity of 10 cP. Thus, under physiological conditions of increased viscosity the NADH association may be significantly affected by diffusion effects. In order to estimate accurately the maximum diffusion-controlled rate constant from diffusion theory, the diffusion coefficients of NADH were measured in poly(ethylene glycol)/water mixtures and were found to vary inversely as the solvent viscosity raised to the power of 0.5. The non-Stokesian behaviour of molecules as large as NADH in polymer/water mixtures may be a serious limitation to the routine use of poly(ethylene glycol) as a viscosogen for diffusion studies.

Pig heart fumarase really does exhibit negative kinetic co-operativity at a constant ionic strength.

The kinetics of the action of fumarase on L-malate and fumarate were investigated at constant ionic strength. This was done to evaluate reports that fumarase follows simple Michaelis-Menten kinetics. However, when pH, buffer concentration and ionic strength are all maintained at constant values, the Lineweaver-Burk plots exhibit pronounced downward curvature, characteristic of negative kinetic co-operativity.