Real-world treatment of metastatic hormone-sensitive prostate cancer in the USA, Europe and Asia.

Aim: To characterize real-world patients with metastatic hormone-sensitive prostate cancer (mHSPC) and treating physicians and evaluate treatment trends and baseline concordance versus guidelines internationally. Materials & methods: Retrospective, cross-sectional data from the Ipsos Global Oncology Monitor database 2018-2020 were used for descriptive analysis of mHSPC patients, treating physicians and treatment utilization. Results: Among the 6198 mHSPC patients from five countries, the most common treatment was either androgen deprivation therapy (ADT) monotherapy or first-generation androgen receptor inhibitor + ADT. Second-generation androgen receptor inhibitor use was only initiating but increasing over the study period. Conclusion: Despite contemporaneous guidelines recommending treatment intensification of ADT in combination with novel antihormonals or docetaxel, 76.1% of reported mHSPC patients received non-guideline-concordant care.

Prostate cancer is the second most common cancer among men worldwide and a leading cause of cancer-related death globally. Metastatic hormone-sensitive prostate cancer (mHSPC) refers to the stage of prostate cancer where it has spread to other parts of the body ('metastatic') but still responds to hormonal therapy ('hormone-sensitive'), such as androgen deprivation therapy (ADT). Treatment guidelines around the world for men with mHSPC have changed over time, but there remains a lack of understanding of how well guidelines are followed in real-world practice. Consequently, this study analyzes real-world data from five countries between 2018 and 2020 to understand treatment patterns, baseline concordance versus guidelines and potential drivers of treatment trends. The study found prevalent use of ADT monotherapy and older antihormonal agents, and only marginal but increasing use of novel antihormonals in real-world practice. These practices deviate from guidelines from the study period, which generally recommended ADT combination with either newer antihormonal agents or docetaxel for patients with mHSPC. Overall, the proportion of the 6198 patients treated with non­guideline-concordant therapies was 76.1%. Since guideline-recommended care is associated with better outcomes, this baselining finding highlights the need for appropriate treatment selection and intensification for mHSPC patients.

The Hospitalization-Related Costs of Adverse Events for Novel Androgen Receptor Inhibitors in Non-Metastatic Castration-Resistant Prostate Cancer: An Indirect Comparison.

INTRODUCTION: Three novel androgen receptor inhibitors are approved in the USA for the treatment of non-metastatic castration-resistant prostate cancer (nmCRPC): apalutamide, enzalutamide, and darolutamide. All three therapies have demonstrated prolonged metastasis-free survival in their respective phase III trials, with differing safety profiles. The objective of this study was to compare the mean per-patient costs of all-cause adverse events (AEs) requiring hospitalization between darolutamide versus apalutamide and enzalutamide for nmCRPC in the USA. METHODS: All-cause grade ≥ 3 AEs with corresponding any-grade AEs reported among at least 10% of patients in any arm of the ARAMIS (darolutamide), SPARTAN (apalutamide), and PROSPER (enzalutamide) trials were selected for inclusion in the primary analyses. After matching-adjusted indirect comparison, AE costs were calculated by multiplying the AE rates from the trials by their respective unit costs of hospitalization taken from the US Healthcare Cost and Utilization Project (HCUP) database. Sensitivity analyses which further included any-grade AEs reported among at least 5% of patients were also performed. RESULTS: After reweighting and adjusting for the trials' placebo arms, the mean per-patient AE costs were $1021 and $387 lower for darolutamide than for apalutamide and enzalutamide, respectively, over the trials' duration (SPARTAN and PROSPER, 43 months; ARAMIS, 48 months). For darolutamide vs. apalutamide, the largest drivers of the per-patient cost differences were fracture (adjusted difference $416), hypertension ($143), and rash ($219); for darolutamide vs. enzalutamide, they were fatigue not including asthenia ($290) and hypertension including increased blood pressure (i.e., any AE of hypertension or with elevated blood pressure not yet classified as hypertension) ($60). The results of the sensitivity analyses were consistent with the primary results. CONCLUSIONS: Patients with nmCRPC treated with darolutamide in ARAMIS incurred lower AE-related costs (USD), as determined using HCUP costing data, compared with patients treated with either apalutamide (in SPARTAN) or enzalutamide (in PROSPER).

Indirect Comparison of Darolutamide versus Apalutamide and Enzalutamide for Nonmetastatic Castration-Resistant Prostate Cancer.

PURPOSE: No published head-to-head randomized trials have compared the safety and efficacy of darolutamide vs apalutamide or enzalutamide in nonmetastatic castration-resistant prostate cancer. This study compares prespecified adverse events and metastasis-free survival associated with darolutamide vs apalutamide, and darolutamide vs enzalutamide, via matching-adjusted indirect comparisons. MATERIALS AND METHODS: Individual patient data from the phase III ARAMIS trial (NPLACEBO=553; NDAROLUTAMIDE=943) were selected and reweighted to match the inclusion criteria and baseline characteristics published for the phase III SPARTAN (NPLACEBO=401; NAPALUTAMIDE=806) and PROSPER (NPLACEBO=468; NENZALUTAMIDE=933) trials. Only baseline factors consistently reported across trials were included as matching covariates. Both indirect comparisons matched on age, prostate specific antigen level and doubling time, Eastern Cooperative Oncology Group performance status, Gleason score, and bone-sparing agent use. Darolutamide vs apalutamide also matched on prior surgery and darolutamide vs enzalutamide also matched on region. Risk differences and odds ratios were calculated for adverse events and hazard ratios for metastasis-free survival. RESULTS: No differences in metastasis-free survival hazard ratios were found after matching in either comparison. However, fall, fracture and rash rates were statistically significantly lower in favor of darolutamide vs apalutamide. Fall, dizziness, mental impairment, fatigue and severe fatigue rates were statistically significantly lower in favor of darolutamide vs enzalutamide. CONCLUSIONS: While metastasis-free survival did not differ across drugs in these cross-trial indirect comparisons, darolutamide showed a favorable safety and tolerability profile in prespecified adverse events vs apalutamide and enzalutamide. Consideration of these adverse events is important in clinical decision-making and treatment selection in nonmetastatic castration-resistant prostate cancer.

Patient and caregiver benefit-risk preferences for nonmetastatic castration-resistant prostate cancer treatment.

BACKGROUND: Recently approved second-generation androgen receptor inhibitors (SGARIs) for non-metastatic castration-resistant prostate cancer (nmCRPC) have similar efficacy but differ in safety profiles. We used a discrete choice experiment (DCE) to examine how nmCRPC patients and caregivers perceive the benefits versus risks of these new treatments. METHODS: An online DCE survey with 14 treatment choice questions was administered to nmCRPC patients and caregivers. Each choice question compared two hypothetical medication profiles varying in terms of 5 safety attributes (risk or severity of adverse events [AEs]: fatigue, skin rash, cognitive problems, serious fall, and serious fracture) and two efficacy attributes (duration of overall survival [OS] and time to pain progression). Random parameters logit models were used to estimate each attribute's relative importance. We also estimated the amounts of OS that respondents were willing to forego for a reduction in AEs. RESULTS: In total, 143 nmCRPC patients and 149 caregivers viewed the AEs in following order of importance (most to least): serious fracture, serious fall, cognitive problems, fatigue, and skin rash. On average, patients were willing to trade 5.8 and 4.0 months of OS to reduce the risk of serious fracture and fall, respectively, from 3% to 0%; caregivers were willing to trade 6.6 and 5.4 months of OS. CONCLUSIONS: nmCRPC patients and caregivers preferred treatments with lower AE burdens and were willing to forego OS to reduce the risk and severity of AEs. Our results highlight the importance of carefully balancing risks and benefits when selecting treatments in this relatively asymptomatic population.

Physician preferences for non-metastatic castration-resistant prostate cancer treatment.

BACKGROUND: Recent approvals of second-generation androgen receptor inhibitors (SGARIs) have changed the treatment landscape for non-metastatic castration-resistant prostate cancer (nmCRPC). These SGARIs have similar efficacy but differ in safety profiles. We used a discrete choice experiment to explore how United States physicians make treatment decisions between adverse events (AEs) and survival gains in nmCRPC, a largely asymptomatic disease. METHODS: Treating physicians (n = 149) participated in an online survey that included 14 treatment choice questions, each comparing 2 hypothetical treatment profiles, which varied in terms of 5 safety and 2 efficacy attributes. We described safety attributes (fatigue, skin rash, cognitive problems, falls, and fractures) in terms of severity and frequency, and efficacy attributes (overall survival [OS] and time to pain progression) in terms of duration of effect. We used a random parameters logit model to estimate preference weights and importance scores for each attribute. We also estimated the amount of survival gain physicians were willing to trade for a reduction in specific AEs between treatment options. RESULTS: Physicians placed more importance on survival than on time to pain progression, and viewed a reduction in cognitive problems from severe to none, a reduction in risk of a serious fracture from 8% to none, and a reduction in fatigue from severe to none as the most important safety attributes. Physicians were willing to forego 9.1 and 6.6 months of OS, respectively, to reduce cognitive problems and fatigue from severe to mild-to-moderate. To reduce the risk of a serious fracture from 8 to 5% and 5% to none, physicians were willing to trade 3.9 and 5.3 months of OS, respectively. CONCLUSIONS: Physicians were willing to trade substantial amounts of survival to avoid AEs between hypothetical treatments. These results emphasize the importance of carefully balancing therapies' benefits and risks to ultimately optimize the overall quality of nmCRPC patients' survival. Nonetheless, it is noted that the results from the study sample of 149 physicans may not be representative of the viewpoints of all nmCRPC-treating physicians.

Oxygen isotope effects during microbial sulfate reduction: applications to sediment cell abundances.

The majority of anaerobic biogeochemical cycling occurs within marine sediments. To understand these processes, quantifying the distribution of active cells and gross metabolic activity is essential. We present an isotope model rooted in thermodynamics to draw quantitative links between cell-specific sulfate reduction rates and active sedimentary cell abundances. This model is calibrated using data from a series of continuous culture experiments with two strains of sulfate reducing bacteria (freshwater bacterium Desulfovibrio vulgaris strain Hildenborough, and marine bacterium Desulfovibrio alaskensis strain G-20) grown on lactate across a range of metabolic rates and ambient sulfate concentrations. We use a combination of experimental sulfate oxygen isotope data and nonlinear regression fitting tools to solve for unknown kinetic, step-specific oxygen isotope effects. This approach enables identification of key isotopic reactions within the metabolic pathway, and defines a new, calibrated framework for understanding oxygen isotope variability in sulfate. This approach is then combined with porewater sulfate/sulfide concentration data and diagenetic modeling to reproduce measured 18O/16O in porewater sulfate. From here, we infer cell-specific sulfate reduction rates and predict abundance of active cells of sulfate reducing bacteria, the result of which is consistent with direct biological measurements.

Direct medical costs of treatment in newly-diagnosed high-grade glioma among commercially insured US patients.

AIM: This analysis assessed the direct medical costs of newly-diagnosed, temozolomide (TMZ)-treated glioblastoma (GBM) from the perspective of a US commercial setting. MATERIALS AND METHODS: The analysis included subjects identified from the IMS PharMetrics LifeLink Plus™ claims database from January 1, 2008 to August 31, 2014 who were ≥18 years of age, had ≥1 malignant brain cancer diagnosis, had brain surgery ≤90 days prior to TMZ initiation, had TMZ treatment, and were continuously enrolled for ≥12 months pre-diagnosis and ≥1 month post-diagnosis. Per-patient per-month (PPPM) and cumulative costs from 3 months pre-diagnosis to various post-diagnosis follow-up time points were calculated. Multivariable analyses were used to estimate adjusted mean cost and identify contributors of cost. RESULTS: The study included 2,921 subjects (median age = 56 years; 60% male). After diagnosis, the median (interquartile range, IQR) number of inpatient, emergency department, and outpatient visits were 2 (1-4), 1 (1-3), and 19 (13-27); median (IQR) length of stay per hospitalization was 5 (3-9) days. Mean total cumulative costs per patient from 3 months pre-diagnosis to 12 months and to 5 years post-diagnosis were $201,749 (197,490-206,024) and $268,031 (262,877-274,416). Mean (SD) PPPM costs were $818 (1,128) and $7,394 (8,676) pre- and post-GBM diagnosis, respectively. The variables most predictive of cumulative costs included radiation therapy (+$81,732), ≥2 weeks of hospitalization (+$49,629), and ≥7 MRI scans (+$40,105). CONCLUSIONS: The direct medical costs of newly-diagnosed, TMZ-treated GBM in commercially insured patients are substantial, with estimated total cumulative costs of $268,031.

The Importance of Economic Perspective and Quantitative Approaches in Oncology Value Frameworks of Drug Selection and Shared Decision Making.

The debate around value in oncology drug selection has been prominent in recent years, and several professional bodies have furthered this debate by advocating for so-called value frameworks. Herein, we provide a viewpoint on these value frameworks, emphasizing the need to consider 4 key aspects: (1) the economic underpinnings of value; (2) the importance of the perspective adopted in the valuation; (3) the importance of the difference between absolute and relative measures of risk and measuring patient preferences; and (4) the recognition of multiple quality-of-life (QoL) domains, and the aggregation and valuation of those domains, through utilities within a multicriteria decision analysis, may allow prioritization of QoL above the tallying of safety events, particularly in a value framework focusing on the individual patient. While several frameworks exist, they incorporate different attributes and-importantly-assess value from alternative perspectives, including those of patients, regulators, payers, and society. The various perspectives necessarily lead to potentially different, if not sometimes divergent, conclusions about the valuation. We show that the perspective of the valuation affects the framing of the risk/benefit question and the methodology to measure the individual patient choice, or preference, as opposed to the collective, or population, choice. We focus specifically on the American Society of Clinical Oncology (ASCO) Value Framework. We argue that its laudable intent to assist in shared clinician-patient decision making can be augmented by more formally adopting methodology underpinned by micro- and health economic concepts, as well as application of formal quantitative approaches. Our recommendations for value frameworks focusing on the individual patient, such as the ASCO Value Framework, are 3-fold: (1) ensure that stakeholders understand the importance of the adopted (economic) perspective; (2) consider using exclusively absolute measures of risk and formal patient-preference methodology; and (3) consider foregoing safety parameters for higher-order utility considerations. DISCLOSURES: No funding was received for conceptualizing, writing, and/or editing this manuscript. Waldeck and White are employees of, and received stock option grants from, Celldex Therapeutics. Van Hout and Botteman are employees and shareholders of Pharmerit International. Pharmerit International is a research contractor for Celldex. All authors have retained editorial control of the content of the manuscript. Conceptualization of this viewpoint article was contributed primarily by Waldeck, along with Botteman, White, and van Hout. Data analysis and revision of the manuscript was contributed equally by all the authors. The manuscript was written by Waldeck, Botteman, van Hout, and White.

Bis-phenanthroline derivatives as suitable scaffolds for effective G-quadruplex recognition.

Selective recognition of DNA folding is central to multiple biological and pharmacological applications aimed at precise targeting of distinct genomic regions. Here, we focused on the recognition of physiologically relevant G-quadruplex (G-4) structures by bis-phenanthroline (bis-Phen) ligands containing two Phen moieties covalently linked through an amine or thioether bond. The transition metal ions Mn(2+), Ni(2+), Cu(2+), and the biologically relevant Mg(2+) and Zn(2+) efficiently form 1 : 1 bis-Phen complexes characterised by a large planar structure fit to successfully recognise G-quartet arrangements.Interestingly, metal ion complexation dramatically affects ligand-stabilising effects on G-quadruplex, the melting temperature of the folded structure being increased up to 30 degrees C at ligand concentrations as low as 1 microM in the presence of Ni(2+) and Cu(2+). In addition, the test complexes were able to induce G-4 formation from essentially unfolded G-rich sequences even in the absence of K(+) ions as shown by gel shift and circular dichroism experiments. In line with their G-4 stabilising properties bis-Phen complexes are effective inhibitors of telomerase activity, Ni(II) complexes being effective in the sub-micromolar range. This is combined with lack of unselective DNA-damaging activity and short-term cellular toxicity, which makes the novel compounds (above all their Ni(II) complexes) interesting antiproliferative drug leads.

[Anomalies of the skull in cleidocranial dysplasia]. / Schädelanomalien bei Dysostosis cleidocranialis. Eine HNO-ärztliche und kraniofaziale Röntgenstudie.

BACKGROUND: The description of the otorhinolaryngeal and craniofacial anomalies in patients with cleidocranial dysplasia. METHODS: For this study, 26 patients with cleidocranial dysplasia were examined after their medical history had been recorded. The main focus was placed on otorhinolaryngological and orthodontic findings. RESULTS: The portion of spontaneous mutations in our patient population was 46.1%. All patients exhibited otorhinolaryngological and craniofacial anomalies. While single ENT-anomalies were expressed in 76.9%-92.3% of the patients, the craniofacial findings were distributed over 84.6%-92.3%. CONCLUSION: The expression of this rare disorder is variable and its symptomatology not always distinct. Otorhinolaryngological and craniofacial anomalies are often apparent. Appropriate treatment can significantly contribute to an improvement in the patient's quality of life. In cases of ambiguous findings, we recommend consultation with an experienced clinician as well as genetic counselling.

[How do metallic middle ear implants behave in the MRI?]. / Wie verhalten sich metallhaltige Mittelohrimplantate in der Kernspintomographie?

BACKGROUND: Magnetic resonance imaging (MRI) has gained in frequency and importance as a diagnostic procedure. In respect to the close anatomical relationship in the temporal bone it is necessary to know whether it is hazardous to patients with metallic middle ear implants regarding displacement and rise in temperature. For the MR image quality artefacts caused by metallic prostheses should be low. METHODS: Four different stapes prostheses made from titanium, gold, teflon/platinum and teflon/steel, a titanium total ossicular reconstruction prosthesis (TORP) and two ventilation tubes (made from titanium and gold) were tested in a 1.5 Tesla MRI machine regarding their displacement. All objects were first placed in a petri dish, then suspended from a thread and finally immersed in a dish filled with Gadolinium. Temperature changes of the implants were recorded by a pyrometer. RESULTS: None of the implants moved when they were placed in the petri dish or suspended from the thread. On the water surface the teflon/platinum and the teflon/steel pistons adjusted their direction with their axis longitudinally to the MRI scanner opening and the teflon/steel piston floated towards the MRI-machine when put close enough to the scanner opening. No rise in temperature was recorded. All implants showed as little artefacts that would still make an evaluation of the surrounding tissue possible. CONCLUSION: Patients with any of the metallic middle ear implants that were examined in this study may undergo MRI-investigations without significant adverse effects.

Value of high-resolution MR in patients scheduled for cochlear implantation.

OBJECTIVE: To determine sensitivity and specificity of high-resolution MR imaging and of high-resolution axial CT (HRCT) and to compare the two modalities in predicting the surgical and functional success of cochlear implantation. MATERIAL AND METHODS: The presurgical MR images (2D T2W TSE, 3D T2*W CISS, plain and contrast-enhanced 3D T1W MP-RAGE) and axial HRCT findings of 26 patients were evaluated with regard to the predictive value concerning the success of cochlear implantation. RESULTS: We found a high correlation between MR and HRCT and the success of cochlear implantation. In all 26 patients, the MR-based predictions concerning the success of cochlear implantation were correct. In 10 patients, MR gave additional information to HRCT. In all patients, MR gave sufficient information about the status of the inner ear, inner auditory canal and cochlear nerve to aid the surgeon during the operation. CONCLUSION: A high-resolution MR protocol consisting of coronal 2D T2W TSE, 3D T2*W axial CISS, plain and contrast-enhanced sagittal T1W 3D MP-RAGE is recommended for the evaluation of candidates scheduled for cochlear implantation. It provides information which cannot be obtained by HRCT.

Comparison of a T2* w. 3D CISS and a T2 w. 3D turbo spin echo sequence for the anatomical study of facial and vestibulocochlear nerves.

Thirty healthy volunteers were examined with a T2* w. 3D CISS and a T2 w. 3D turbo spin echo (TSE) sequence in order to compare the facial and vestibulocochlear nerve detectability in the cerebellopontine angle and the internal auditory canal. CISS was significantly better than 3D TSE for nerve detectability in the cerebellopontine angle and equally as good as 3D TSE in the internal auditory canal. We would therefore recommend the inclusion of CISS in an MR imaging protocol of the facial and vestibulocochlear nerves.

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans.

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

[3D CISS, 3D MP-PAGE and 2D TSE for the preoperative MRI prior to cochlear implantation]. / 3D CISS, 3D MP-RAGE und 2D TSE für die präoperative MRT vor Cochlea Implant.

PURPOSE: The aim of this study was to determine the presurgical predictive value of high resolution MRI in patients scheduled for cochlear implantation. METHOD AND MATERIAL: The presurgical MRI (3D CISS, 3D MP-RAGE with and without i.v. contrast medium, 2D TSE) findings of 54 patients and the intraoperative situation reported by the surgeon were compared retrospectively. The surgical and functional success of the cochlear implantation was evaluated. RESULTS: We found a high degree of correlation between MRI and intraoperative findings concerning the patency of the whole cochlea and anomalies as well as in the diagnosis of pathology of the cochlear, vestibular and facial nerves and in anomalies of the internal auditory canal. However, in four out of 54 patients there was a false negative prediction regarding the patency of the cochlea. The sensitivity was 50% (4/8), the specificity 100% (46/46). Concerning the surgical success the accuracy was 100%. In all patients MRI gave sufficient anatomical information to the surgeon concerning the jugular bulb and the facial nerve. CONCLUSION: A high-resolution MRI protocol consisting of coronal 2D T2w TSE, 3D T2*w transverse CISS; plain and contrast enhanced sagittal T1w 3D MP-RAGE is recommended for the evaluation of candidates scheduled for cochlear implantation.

NMR measurements of Ca2+ and H+ transport mediated by A23187 and reconstituted plasma membrane Ca(2+)-ATPase.

NMR-based assays for measuring the fluxes of Ca2+, H+, and ATP in liposomal systems are presented. The 19F NMR Ca(2+)-chelating molecule 5,5-difluoro-1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) was trapped inside large unilamellar vesicles and used to monitor passive and A23187-mediated Ca2+ transport into them. The data were analyzed using progress curves of the transport reaction. They demonstrated the general applicability of 5FBAPTA as a 19F NMR probe of active Ca2+ transport. 31P NMR time-courses were used to monitor simultaneously the ATP hydrolysing activity of the reconstituted human erythrocyte Ca(2+)-ATPase and the concomitant acidification of the reaction medium in a suspension of small unilamellar vesicles. Using an estimate of the extraliposomal buffering capacity, the H+/ATP coupling stoichiometry, in the presence of A23187, was estimated from the NMR-derived data at steady state; it amounted to 1.4 +/- 0.3. This result is discussed with respect to the issue of molecular 'slip' in the context of a non-equilibrium thermodynamics model of the pump (accompanying paper in this issue). Importantly, NMR, in contrast to optical detection methods, can potentially register all fluxes and (electro)chemical gradients involved in the Ca(2+)-ATPase-mediated H+/Ca2+ counterport, in a single experiment.

A non-equilibrium thermodynamics model of reconstituted Ca(2+)-ATPase.

A non-equilibrium thermodynamics (NET) model describing the action of completely coupled or 'slipping' reconstituted Ca(2+)-ATPase is presented. Variation of the coupling stoichiometries with the magnitude of the electrochemical gradients, as the ATPase hydrolyzes ATP, is an indication of molecular slip. However, the Ca2+ and H+ membrane-leak conductances may also be a function of their respective gradients. Such non-ohmic leak typically yields 'flow-force' relationships that are similar to those that are obtained when the pump slips; hence, caution needs to be exercised when interpreting data of Ca(2+)-ATPase-mediated fluxes that display a non-linear dependence on the electrochemical proton (delta mu H) and/or calcium gradients (delta mu Ca). To address this issue, three experimentally verifiable relationships differentiating between membrane leak and enzymic slip were derived. First, by measuring delta mu H as a function of the rate of ATP hydrolysis by the enzyme. Second, by measuring the overall 'efficiency' of the pump as a function of delta mu H. Third, by measuring the proton ejection rate by the pump as a function of its ATP hydrolysis rate.

Oxidation of high density lipoproteins. II. Evidence for direct reduction of lipid hydroperoxides by methionine residues of apolipoproteins AI and AII.

Human high density lipoproteins (HDL) can reduce cholesteryl ester hydroperoxides to the corresponding hydroxides (Sattler W., Christison J. K., and Stocker, R. (1995) Free Radical Biol. & Med. 18, 421-429). Here we demonstrate that this reducing activity extended to hydroperoxides of phosphatidylcholine, was similar in HDL2 and HDL3, was independent of arylesterase and lecithin:cholesteryl acyltransferase activity, was unaffected by sulfhydryl reagents, and was expressed by reconstituted particles containing apoAI or apoAII only, as well as isolated human apoAI. Concomitant with the reduction of lipid hydroperoxides specific oxidized forms of apoAI and apoAII formed in blood-derived and reconstituted HDL. Similarly, specific oxidized forms of apoAI accumulated upon treatment of isolated apoAI with authentic cholesteryl linoleate hydroperoxide. These specific oxidized forms of apoAI and apoAII have been shown previously to contain Met sulfoxide (Met(O)) at Met residues and are also formed when HDL is exposed to Cu2+ or soybean lipoxygenase. Lipid hydroperoxide reduction and the associated formation of specific oxidized forms of apoAI and apoAII were inhibited by solubilizing HDL with SDS or by pretreatment of HDL with chloramine T. The inhibitory effect of chloramine T was dose-dependent and accompanied by the conversion of specific Met residues of apoAI and apoAII into Met(O). Canine HDL, which contains apoAI as the predominant apolipoprotein and which lacks the oxidation-sensitive Met residues Met112 and Met148, showed much weaker lipid hydroperoxide reducing activity and lower extents of formation of oxidized forms of apoAI than human HDL. We conclude that the oxidation of specific Met residues of apoAI and apoAII to Met(O) plays a significant role in the 2-electron reduction of hydroperoxides of cholesteryl esters and phosphatidylcholine associated with human HDL.

Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that accompanies lipid peroxidation and can be enhanced by alpha-tocopherol.

The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL's alpha-tocopherol (alpha-TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of alpha-TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with alpha-TOH resulted in a dose-dependent increase in the extent of peroxyl radical-induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0. 92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). alpha-TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, alpha-TOH can promote this process.

Electron paramagnetic resonance studies of succinate:ubiquinone oxidoreductase from Paracoccus denitrificans. Evidence for a magnetic interaction between the 3Fe-4S cluster and cytochrome b.

Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic "signatures" accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochrome b, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4-10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate- and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity (