Tunneling in tocopherol-mediated peroxidation of 7-dehydrocholesterol.

The peroxidation of 7-dehydrocholesterol (7-DHC), a biosynthetic precursor to vitamin D3 and cholesterol, has been linked to the pathophysiology of Smith-Lemli-Optiz syndrome (SLOS), a devastating human disorder. In SLOS, 7-DHC plasma and tissue levels are elevated because of defects in the enzyme that convert it to cholesterol. α-Tocopherol can mediate the peroxidation of 7-DHC under certain circumstances and this prompted us to investigate the kinetic isotope effect (KIE) during this process. Thus, 9,14-d2-7-DHC was synthesized using a photochemical cyclization of deuterium-reinforced previtamin D3 (retro to its biosynthesis). Subsequently, we carried out co-oxidation of 9,14-h2-25,26,26,26,27,27,27-d7- and 9,14-d2-7-DHC in the presence of α-tocopherol under conditions that favor TMP. By monitoring the products formed from each precursor using mass spectrometry, the KIE for the hydrogen (deuterium) atom removal at C9 was found to be 21 ± 1. This large KIE value indicates that tunneling plays a role in the hydrogen atom transfer step in the tocopherol-mediated peroxidation of 7-DHC.

MRI of inflammatory spondyloarthropathy following traumatic cauda equina syndrome.

BACKGROUND CONTEXT: Spondyloarthropathy has been described radiographically in patients following paralysis from spinal cord trauma. Onset of these findings after cauda equina syndrome have not been reported previously. Furthermore, the magnetic resonance documentation of its early evolution has not been recorded. PURPOSE: We report a case of early-onset spondyloarthropathy shown by magnetic resonance imaging (MRI) in a patient with cauda equina syndrome due to bilateral sacral insufficiency fractures. STUDY DESIGN: Unique case study review, one case. METHODS: Review of the clinical case notes and imaging including initial and subsequent MR imaging. RESULTS: The initial MRI of the lumbosacral spine showed bilateral sacral insufficiency fractures with a kyphotic deformity. The vertebral bodies were normal on the initial computed tomography and MRI studies, which did not reveal pre-existing features of sacroiliitis. The second MRI performed 5 months later clearly showed spondylitis at multiple vertebral levels with partial resolution 18 months post injury. CONCLUSION: Spondyloarthropathy in patients with paralysis due to spinal cord injury is well documented in the English language literature, but until now this has not been demonstrated by MRI. It is a rare complication of traumatic cauda equina syndrome that commences soon after the traumatic event and can resolve spontaneously.

A new method for measurement of subcoracoid outlet and its relationship to rotator cuff pathology at MR arthrography.

OBJECTIVE: Orthopaedic surgical studies have shown that variations in the vertical distance between the tip of the coracoid process and the supra-glenoid tubercle alter the shape of the subcoracoid outlet. Our objective was to measure the vertical distance between the coracoid tip and the supra-glenoid tubercle (CTGT) on MR and to assess whether this showed better correlation with rotator cuff pathology compared with the axial coraco-humeral distance. MATERIALS AND METHODS: A retrospective review was performed of 100 consecutive shoulder MR arthrograms. Vertical distance between the coracoid tip and the supraglenoid tubercle was measured in the sagittal oblique plane. Separate assessment was then made of tendon pathology of the subscapularis, supraspinatus and long head of biceps tendons. Axial coraco-humeral distance was then measured. Correlation between tendon abnormalities and the two measurements was then made. RESULTS: Of the 100 cases, 42 had subscapularis tendon lesions, 21 had lesions of the long head of biceps and 53 had supraspinatus tendon lesions. Mean vertical distance from the coracoid tip to supraglenoid tubercle was greater in those with lesions of any of these tendons and was statistically significant for the supraspinatus group (P = 0.005). Reduced axial coraco-humeral distance was also seen in patients with tendinopathy, although with less statistically significant difference (p = 0.059). CONCLUSION: Our results support orthopaedic studies that have shown that the vertical distance between the coracoid tip and the supraglenoid tubercle increases the incidence and risk of rotator cuff disease by altering the shape of the subcoracoid outlet.

Removal and detoxification of pentahalogenated phenols using a photocatalytically induced enzymatic process.

Poly-halogenated phenols generated from a range of industrial processes can find their way into rivers and ground water. Here we report on a potential treatment for reducing the toxicity of these aqueous pollutants using two highly toxic penta-halogenated phenols (pentachlorophenol (PCP) and pentabromophenol (PBP)) as surrogates. Solutions were passed through a glass column packed with a silica support fused with titanium dioxide (TiO2) and horseradish peroxidase (HRP) immobilized on its TiO2/glass surface (HRP-Tglass). TiO2 photocatalysis was activated through irradiation with UVB (320 nm) which in turn activated the HRP. Two operational flow rates (0.5 and 1.25 mL min-1; hydraulic retention times (HRTs) of 20 and 8 min, respectively), tested the effect of retention time on the extent of degradation and reduction in toxicity of the treated effluent. Microtox® was used to measure the toxicity of the substrate and its by-products at both flow rates. At the highest flow rate, dehalogenation was limited (removal of 37 % chlorine and 22 % bromine) and the toxicity of the reaction products increased. At the lowest flow rate, the longer exposure time resulted in approximately 97 % and 96 % transformation of PCP and PBP, respectively, a greater degree of dehalogenation (removal of 65 % chlorine and 70 % bromine) and a substantial decrease in toxicity of the treated solutions. The higher toxicity of effluent from the higher flow rate was attributed to the initial degradation products being more toxic than the substrates. With a longer HRT, these were then further broken down to less toxic products. Additional toxicity tests (Hydra hexactinella (Hydra) and Chinese Hamster Ovary (CHO) cell toxicity were conducted on the effluent from the lowest flow rate. Both were less sensitive than the Microtox test, with Hydra proving more sensitive than CHO. The novelty of this work is the toxicity risk assessment of the products resulting from the use of a spatially separated immobilized enzyme and photooxidation system. The system was robust and showed no decrease in treatment efficacy over 10 h.

Selective gamma-ketoaldehyde scavengers protect Nav1.5 from oxidant-induced inactivation.

The cardiac sodium channel (SCN5A, Na(V)1.5) is a key determinant of electrical impulse conduction in cardiac tissue. Acute myocardial infarction leads to diminished sodium channel availability, both because of decreased channel expression and because of greater inactivation of channels already present. Myocardial infarction leads to significant increases in reactive oxygen species and their downstream effectors including lipoxidation products. The effects of reactive oxygen species on Na(V)1.5 function in whole hearts can be modeled in cultured myocytes, where oxidants shift the availability curve of I(Na) to hyperpolarized potentials, decreasing cardiac sodium current at the normal activation threshold. We recently examined potential mediators of the oxidant-induced inactivation and found that one specific lipoxidation product, the isoketals, recapitulated the effects of oxidant on sodium currents. Isoketals are highly reactive gamma-ketoaldehydes formed by the peroxidation of arachidonic acid that covalently modify the lysine residues of proteins. We now confirm that exposure to oxidants induces lipoxidative modification of Na(V)1.5 and that the selective isoketal scavengers block voltage-dependent changes in sodium current by the oxidant tert-butylhydroperoxide, both in cells heterologously expressing Na(V)1.5 and in a mouse cardiac myocyte cell line (HL-1). Thus, inhibition of this lipoxidative modification pathway is sufficient to protect the sodium channel from oxidant induced inactivation and suggests the potential use of isoketal scavengers as novel therapeutics to prevent arrhythmogenesis during myocardial infarction.

Continuous enzymatic treatment of 4-bromophenol initiated by UV irradiation.

Horseradish peroxidase (HRP) can be used for the treatment of halogenated phenolic substances. In the presence of hydrogen peroxide phenols are oxidized to form polymers which undergo partial dehalogenation. However, when immobilized, the peroxidase is subject to inactivation due to blockage of the active sites by the growing polymers and to deactivation by elevated levels of hydrogen peroxide. When HRP immobilized on a novel glass-based support incorporating titanium dioxide is subjected to UV irradiation, hydrogen peroxide is produced and the nascent polymer is removed. In this work a reactor was constructed that utilized HRP immobilized on the novel support and the in situ production of hydrogen peroxide to treat 4-bromophenol as a model substrate. The system was operated for almost 17 hours with no apparent decline in activity.

Variations in color and reflectance on the surface of asteroid (101955) Bennu.

Visible-wavelength color and reflectance provide information about the geologic history of planetary surfaces. Here we present multispectral images (0.44 to 0.89 micrometers) of near-Earth asteroid (101955) Bennu. The surface has variable colors overlain on a moderately blue global terrain. Two primary boulder types are distinguishable by their reflectance and texture. Space weathering of Bennu surface materials does not simply progress from red to blue (or vice versa). Instead, freshly exposed, redder surfaces initially brighten in the near-ultraviolet region (i.e., become bluer at shorter wavelengths), then brighten in the visible to near-infrared region, leading to Bennu's moderately blue average color. Craters indicate that the time scale of these color changes is ~105 years. We attribute the reflectance and color variation to a combination of primordial heterogeneity and varying exposure ages.

Oxidative stress, serotonergic changes and decreased ultrasonic vocalizations in a mouse model of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome is an inherited monogenic disorder in which mutations to the 7-dehydrocholesterol (7-DHC) reductase (Dhcr7) gene lead to deficits in cholesterol synthesis. As a result, many patients suffer from gross physiological and neurological deficits. The purpose of this study was to identify a potential abnormal behavioral phenotype in a compound mutant mouse model for Smith-Lemli-Opitz disease (Dhcr7 Δ3-5/T93M ) to further validate the model and to provide potential targets for future therapeutic interventions. We also sought to identify some of the underlying changes in brain function that may be responsible for behavioral differences among groups. The Dhcr7 compound mutant mice were smaller than their single mutant littermates. Both single and compound heterozygous mice made fewer ultrasonic vocalizations when separated from the dam, which may suggest a communication deficit in these animals. Striking increases of the highly oxidizable 7-DHC were observed in the compound mutant mice. 7-Dehydrocholesterol is the precursor to cholesterol and builds up because of decreased function of the mutated Dhcr7 enzyme. Additionally, several differences were noted in the serotonergic system including increased expression of the serotonin transporter and increased uptake of serotonin by isolated synaptosomes. We propose that changes to the oxidative environment during development can have a significant impact on the development of serotonergic function and that this contributes to behavioral differences observed in the mutant mice.

Cyclic peroxides and the thiobarbituric assay.

Several monocyclic peroxide compounds and two acyclic hydroperoxides have been tested for activity in the thiobarbituric acid test. All cyclic peroxides tested which have beta dioxygen functionality on non-tertiary carbons gave positive thiobarbituric acid tests at 532 nm. (epsilon = 10(3) --10(4)) Two acyclic unsaturated hydroperoxides which were derived from gamma-linolenic acid also gave positive thiobarbituric acid tests. In addition to the 532-nm-absorbing species, all peroxidic compounds tested showed a transient absorption at 450 nm during the thiobarbituric acid test. The species responsible for this 450 nm absorption appears to be an intermediate in a series reaction sequence. This intermediate is converted, in time, to the 532-nm-absorbing species. Thiobarbituric acid assay of the crude autoxidation product mixture of gamma-linolenic acid also shows this transient 450-nm-absorbing species. Added ferric ion enhances the 532 nm abosrbance of the thiobarbituric acid assay of cyclic peroxides.

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes.

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.

Free radical oxidation of cholesterol and its precursors: Implications in cholesterol biosynthesis disorders.

Free radical oxidation of cholesterol and its precursors contribute significantly to the pathophysiology of a number of human diseases. This review intends to summarize recent developments and provide a perspective on the reactivities of sterols toward free radical oxidation, the free radical reaction mechanism, and the biological consequences of oxysterols derived from the highly oxidizable cholesterol precursor, 7-dehydrocholesterol. We propose that the rigid structures, additional substituents on the double bonds, and the well-aligned reactive C-H bonds in sterols make them more prone to free radical oxidation than their acyclic analogs found in unsaturated fatty acids. The mechanism of sterol peroxidation follows some well-established reaction pathways found in the free radical peroxidation of polyunsaturated fatty acids, but sterols also undergo some reactions that are unique to these compounds. Peroxidation of 7-dehydrocholesterol gives arguably the most diverse set of oxysterol products that have been observed to date. The metabolism of these oxysterols in cells and the biological consequences of their formation will be discussed in the context of the pathophysiology of the human disease Smith-Lemli-Opitz syndrome. Considering the high reactivity of sterols, we propose that a number of other cholesterol biosynthesis disorders may be associated with oxidative stress.

Suggested mechanisms for the production of 4-hydroxy-2-nonenal from the autoxidation of polyunsaturated fatty acids.

Autoxidation of certain polyunsaturated fatty acids (PUFA) produces 4-hydroxy-2-nonenal (HNE). Of all of the many products produced during PUFA autoxidation, HNE is one of the more interesting. It is remarkably cytotoxic, and appears to play a role in certain types of pathology. This manuscript suggests, for the first time, mechanisms that rationalize the production of HNE from PUFA that undergo autoxidation. The mechanisms require, in agreement with the facts, an n-6 fatty acid and iron catalysis.

Chemistry of lipid peroxidation.

The free radical chemistry of lipid peroxidation is complex. The classical mechanism of autoxidation involving a peroxy radical abstracting hydrogen atom from lipid and oxygen addition to the carbon radical thus formed must be modified to include (1) peroxy radical beta fragmentation and (2) peroxy radical cyclization. A host of diene hydroperoxides, cyclic peroxides, bicyclic peroxides and epoxy alcohols may be formed in free fatty acid or phospholipid autoxidation. The distribution of products and the effects of hydrogen atom donors on product distribution are understandable by referring to a general scheme for autoxidation described in Scheme III and in Ref. 10.

In vivo photoactivation of caged-thrombin.

Aberrant ocular neovascularization is a major cause of blindness in the world. Abnormal blood vessels in the eye may produce corneal opacification, corneal transplant rejection, neovascular glaucoma, vitreous hemorrhage, traction retinal detachment, and subretinal scars from choroidal neovascular membranes (1-5). Light-induced clotting of blood within these abnormal vessels could provide a novel method for the ablation of deleterious neovascularization. Thrombin is a serine proteinase that participates in the final stages of the coagulation cascade. An inhibitor of thrombin, p-Amidinophenyl-(E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, MeCINN (1), covalently attaches to the active site serine hydroxyl, inhibiting or caging, the enzyme. Photolysis of the caged-thrombin in vitro causes a trans-cis isomerization of MeCINN which leads to regeneration of active enzyme and cleaving of fibrinogen into fibrin (6). Using a rabbit model of corneal neovascularization, we found that light at 366 nm safely and effectively photoactivates intravenous caged-thrombin and produces localized thrombosis in vivo. These results suggest that intravascular photoactivation of caged-thrombin could be used to occlude abnormal blood vessels in the human eye.

Evaluation of p-amidinophenyl esters as potential antithrombotic agents.

Three p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4]).+ In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 microM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better alpha-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of alpha-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 microliter of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and alpha-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.

Analysis of diacyl peroxides by Ag+ coordination ionspray tandem mass spectrometry: free radical pathways of complex decomposition.

Organic peroxides have significance in organic synthesis and biological processes. Characterization of these compounds with weak O-O bonds is sometimes difficult due to their thermal instability and sensitivity to acid or base. Coordination of diacyl peroxides with AgBF4 provides a means for analysis of these compounds by coordination ionspray tandem mass spectrometry (CIS-MS/MS). Precursor ion (Q1) scans of acetyl benzoyl peroxide give two Ag+ adducts, [M + Ag + solvent]+ and [M + Ag + M]+. These silver ion adducts can be selectively dissociated (CID) to give unique structural information about the analyte. Decomposition of the [M + Ag + solvent]+ adduct generates fragmentation products due to apparent homolytic cleavage of the O-O bond followed by decarboxylation of the resultant radicals. The bis-diacylperoxide complex, [M + Ag + M]+ gives CID pathways that involve homolysis of the (O-O bond and free radical cross-coupling of the two diacyl peroxides coordinated to the silver ion, i.e. formation of dibenzoyl peroxide, phenyl benzoate, and biphenyl from acetyl benzoyl peroxide. The observation of free radical CID modes is uncommon in mass spectrometry but these pathways are consistent with well-known solution and gas phase processes for peroxide compounds. The proposed fragmentation pathways have been supported by experiments with (18)O and deuterated substrates. This technique can be applied to analyze diacyl peroxides with different substituents as well.

Design of unsymmetrical azo initiators to increase radical generation efficiency in low-density lipoproteins.

Lipid peroxidation studies often employ the use of azo initiators to produce a slow, steady source of free radicals, but the lack of initiators capable of efficiently generating radicals in lipid regions has created persistent problems in these investigations. For example, experiments with symmetrical lipophilic or symmetical hydrophilic azo initiators increasingly suggest that their initiation mechanisms in low-density lipoproteins (LDL) rely upon the presence of alpha-tocopherol to mediate peroxidation. We report here the synthesis and study of the new unsymmetrical azo compounds SA-1, SA-2, C-16, C-12, and C-8 that decompose over a range of convenient temperatures and improve radical generation efficiency and access to lipid compartments. The half-life for decomposition (tau(1/2)) of the unsymmetrical initiators at 37 degrees C in methanol covered a range of 121 hours for SA-1, 77 hours for SA-2, and approximately 25 hours for the series C-16, C-12, and C-8. Agarose gel electrophoresis of LDL incubated with these unsymmetrical initiators supports the conclusion that the initiators associate with lipoprotein without disrupting integrity of the particle. The unsymmetical initiator C-8 when compared to symmetical hydrophilic initiator C-0 is capable of providing increased peroxidation of LDL, as monitored by formation of cholesteryl linoleate oxidation products and consumption of alpha-tocopherol. Efficiency of radical generation in lipophilic and hydrophilic compartments was found to be represented with the use of the radical scavenger combination alpha-tocopherol and uric acid, but not with the use of N,N'-Diphenyl-p-phenylenediamine (DPPD) and uric acid. These unsymmetrical initiators, when compared to the widely used symmetrical azo initiators, provide an advantage of free radical production, lipophilic access, and constant radical generation in the investigation of lipid peroxidation in low-density lipoproteins.

Photocoagulation of human plasma: acyl serine proteinase photochemistry.

Human alpha-thrombin or bovine Factor Xa was acylated at the active site serine hydroxyl with alpha-methyl-2-hydroxy-4-diethylaminocinnamic acid. These modified serine proteinase enzymes showed no plasma coagulation biological activity in the absence of light. Photolysis of the acyl serine proteinase enzymes in plasma for 1-35 s with monochromatic 366 nm light isolated from a high pressure mercury arc results in coagulation of the plasma. For example, photolysis of 3 NIH U of the acyl human alpha-thrombin for 5 s in human plasma results in a clot in 23 s. For comparison, 1 NIH U of unmodified human alpha-thrombin gave a clot in 21 s under the conditions of the assay but without photolysis. Appropriate controls showed that the coagulation is the result of the formation of active thrombin due to photodeacylation of the enzymes. The photoinduced clotting time measured is dependent on acyl thrombin concentration and photolysis time. Thus higher concentrations of acyl thrombin and longer photolysis times give a shorter clotting time. A kinetic scheme based upon Lineweaver-Burke analysis of the clotting process is developed.

Synthesis, structure, and thermal properties of 1,2-dipalmitoylgalloylglycerol (DPGG), a novel self-adhering lipid.

A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.

Photo-reversible binding of thrombin to avidin by means of a photolabile inhibitor.

Human alpha-thrombin and Factor Xa were acylated at their active site serine hydroxyls with biotin-substituted cinnamate derivatives 1b-1c. These acyl enzymes (2) showed no enzyme activity in the absence of light. On irradiation with light of wavelength 366 nm for 6 min, however, up to 80% of pre-inhibition activity was regained. This photo-deacylation of the modified enzyme results in the formation of active enzyme and a coumarin by-product (3). In addition, the acyl enzyme that results from incubation of 1c with thrombin was capable of binding to avidin, both immobilized and free in solution. Furthermore, the complex formed between the thrombin acyl enzyme (2c) and avidin was capable of binding to immobilized biotin.