Analysis of serum lysophosphatidylethanolamine levels in patients with non-alcoholic fatty liver disease by liquid chromatography-tandem mass spectrometry.

Lysophosphatidylethanolamines (LysoPEs) are the partial hydrolysis products of phosphatidylethanolamine. Despite the unique in vitro bioactivities of LysoPEs, there are limited reports on the pathophysiological role of LysoPEs in the serum, due to the lack of sensitive analytical methods for determination of each molecular species in clinical samples. Herein, we developed a highly sensitive quantitative method to profile the serum LysoPE species by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM). The internal standard (IS), chemically synthesized in-house, and the lineup of seven major LysoPE species were used in this study. The limits of detection and quantification for each LysoPE species ranged within 0.5-3.3 pmol/mL and 1.0-5.0 pmol/mL, respectively. The combined concentrations of LysoPEs in the serum from healthy subjects (n = 8) and the patients with non-alcoholic fatty liver diseases (NAFLD) including simple steatosis (SS, n = 9) and non-alcoholic steatohepatitis (NASH, n = 27) were 18.030 ± 3.832, 4.867 ± 1.852, and 5.497 ± 2.495 nmol/mL, respectively. The combined and individual concentrations of LysoPEs, except for LysoPE 18:0, significantly decreased in the patients with NAFLD compared with those for the healthy subjects. However, no significant difference was observed between the SS and NASH groups. Our proposed LC-MS/MS method is valid and has advantages of small sample volume, high sensitivity, and simultaneous absolute quantitation for multiple molecular species. This method may enable diagnostic evaluation and elucidation of the as-yet uncovered pathophysiological role of LysoPEs.

Nickel-Mediated Decarbonylation of Simple Unstrained Ketones through the Cleavage of Carbon-Carbon Bonds.

Despite advances in methods for the decarbonylation of aldehydes, the decarbonylation of ketones has been met with limited success because this process requires the activation of two inert carbon-carbon bonds. All of the decarbonylation reactions of simple unstrained ketones reported to date require the addition of a stoichiometric rhodium complex. We report herein the nickel/N-heterocyclic carbene-mediated decarbonylation of simple diaryl ketones. This reaction shows unique acceleration effects based on the presence of both electron-donating and electron-withdrawing groups.

An Efficient Glycoblotting-Based Analysis of Oxidized Lipids in Liposomes and a Lipoprotein.

Although widely occurring lipid oxidation, which is triggered by reactive oxygen species (ROS), produces a variety of oxidized lipids, practical methods to efficiently analyze oxidized lipids remain elusive. Herein, it is shown that the glycoblotting platform can be used to analyze oxidized lipids. Analysis is based on the principle that lipid aldehydes, one of the oxidized lipid species, can be captured selectively, enriched, and detected. Moreover, 3-methyl-1-p-tolyltriazene (MTT) methylates phosphoric and carboxylic acids, and this MTT-mediated methylation is, in combination with conventional tandem mass spectrometry (MS/MS) analysis, an effective method for the structural analysis of oxidized lipids. By using three classes of standards, liposomes, and a lipoprotein, it is demonstrated that glycoblotting represents a powerful approach for focused lipidomics, even in complex macromolecules.

A Conia-Ene-Type Cyclization under Basic Conditions Enables an Efficient Synthesis of (-)-Lycoposerramine†R.

An enantioselective total synthesis of the Lycopodium alkaloid lycoposerramine R is presented. It relies on a base-mediated cyclization that resembles the Conia-ene reaction of ynones and gold-catalyzed variants thereof. Thus, hydrindanones and other functionalized ring systems bearing an exocyclic alkene can be rapidly accessed at room temperature without noble metal catalysis or substrate preactivation.

Nickel/N-Heterocyclic Carbene-Catalyzed Suzuki-Miyaura Type Cross-Coupling of Aryl Carbamates.

The utility of N-heterocyclic carbene ligands in nickel-catalyzed Suzuki-Miyaura cross-coupling of aryl esters and carbamates is investigated. Imidazol-2-ylidene bearing 2-adamantyl groups at its nitrogen atoms generates the most active nickel species among the ligands examined, allowing cross-coupling of a range of aryl carbamates and pivalates. Unlike the previously reported system using tricyclohexylphosphine, this protocol is suitable for the cross-coupling using arylboronic esters in addition to arylboronic acids.

C-H functionalization at sterically congested positions by the platinum-catalyzed borylation of arenes.

Despite significant progress in the area of C-H bond functionalization of arenes, no general method has been reported for the functionalization of C-H bonds at the sterically encumbered positions of simple arenes, such as mesitylene. Herein, we report the development of the first platinum-based catalyst for C-H borylation of arenes and heteroarenes. Notably, this method exhibited high tolerance toward steric hindrance and provided rapid access to a series of 2,6-disubstituted phenylboronic esters, valuable building blocks for further elaborations.

Cation Clock Reactions for the Determination of Relative Reaction Kinetics in Glycosylation Reactions: Applications to Gluco- and Mannopyranosyl Sulfoxide and Trichloroacetimidate Type Donors.

The development of a cation clock method based on the intramolecular Sakurai reaction for probing the concentration dependence of the nucleophile in glycosylation reactions is described. The method is developed for the sulfoxide and trichloroacetimidate glycosylation protocols. The method reveals that O-glycosylation reactions have stronger concentration dependencies than C-glycosylation reactions consistent with a more associative, S(N)2-like character. For the 4,6-O-benzylidene-directed mannosylation reaction a significant difference in concentration dependence is found for the formation of the ß- and α-anomers, suggesting a difference in mechanism and a rationale for the optimization of selectivity regardless of the type of donor employed. In the mannose series the cyclization reaction employed as clock results in the formation of cis and trans-fused oxabicyclo[4,4,0]decanes as products with the latter being strongly indicative of the involvement of a conformationally mobile transient glycosyl oxocarbenium ion. With identical protecting group arrays cyclization in the glucopyranose series is more rapid than in the mannopyranose manifold. The potential application of related clock reactions in other carbenium ion-based branches of organic synthesis is considered.

Prognostic value of tumour-related factors associated with canine retroperitoneal hemangiosarcoma in comparison with other anatomic presentations: A retrospective observational study.

BACKGROUND: Dogs with retroperitoneal hemangiosarcoma (HSA) exhibit variable postoperative median survival times (MST). OBJECTIVE: To retrospectively evaluate the prognostic value of selected tumour-related factors, such as tumour size, rupture, invasion into adjacent tissue, involvement of lymph node and distant metastasis, they were analysed in dogs with retroperitoneal HSA. METHODS: Ten dogs with retroperitoneal HSA managed solely with surgical excision were reviewed and compared with spleen (71) and liver (9) HSA. The Kaplan-Meier method and log-rank analysis were used compare MSTs between factors. Multivariable Cox proportional-hazard analysis was used to compare differences between arising sites. RESULTS: Retroperitoneal HSA showed comparatively longer postoperative MST compared with that of spleen and liver HSA and demonstrated significantly longer MST (p = 0.003) for tumours ≥5 cm (195 days) than <5 cm (70 days). Spleen HSA revealed significantly shorter MSTs in involvement of distant lymph nodes (23 days) and distant metastasis (39 days) than those in negative (83 days, p = 0.002 and 110 days, p < 0.001, respectively). Liver HSA also revealed significantly shorter MST (16.5 days compared with 98 days, p = 0.003) for distant metastasis. Additionally, hazard ratios (HRs) and their forest plot for overall HSA revealed as poor prognostic factors, arising sites (spleen; HR 2.78, p = 0.016 and liver; HR 3.62, p = 0.019), involvement of distant lymph nodes (HR 2.43, p = 0.014), and distant metastasis (HR 2.86, p < 0.001), and as better prognostic factor of tumour size ≥5 cm (HR 0.53, p = 0.037). CONCLUSION: In combination with overall HSA, retroperitoneal HSA shows comparatively longer postoperative MST compared to spleen and liver HSA, associated with tumour size ≥5 cm suggesting better prognostic factor.

Probing the influence of a 4,6-O-acetal on the reactivity of galactopyranosyl donors: verification of the disarming influence of the trans-gauche conformation of C5-C6 bonds.

The effect of a 4,6-O-alkylidene acetal on the rate of acid-catalyzed hydrolysis of methyl galactopyranosides and of spontaneous hydrolysis of 2,4-dinitrophenyl galactopyranosides has been studied through the synthesis and hydrolysis of analogs in which O6 is replaced by a methoxymethylene unit in which the methoxy group adopts either an equatorial or an axial position according to the configuration. Consistent with earlier studies under both acid-catalyzed and spontaneous hydrolysis conditions, the alkylidene acetal, or its 7-carba analog, retards hydrolysis with respect to comparable systems lacking the cyclic protecting group. The configuration at C6 in the 7-carba analogs does not influence the rate of acid-catalyzed hydrolysis but has a minor influence on the rate of spontaneous hydrolysis of the 2,4-dinitrophenyl galactosides, confirming earlier studies on the role played by the hydroxymethyl group conformation on glycoside reactivity. The benzylidene acetal is found to stabilize the α-anomer of galactopyranose derivatives relative to monocyclic analogs. Reasons for the α-selectivity of 4,6-O-benzylidene-protected galactopyranosyl donors bearing neighboring group-active protecting groups at O2 are discussed.

Glycoblotting-based high throughput protocol for the structural characterization of hyaluronan degradation products during enzymatic fragmentation.

Increasing interests have been focused on the functional roles of hyaluronan degradation products, namely hyaluronan oligosaccharides, as signal molecules regulating cell growth, differentiation, malignancy, and inflammatory responses. It is clear that molecular size of hyaluronan oligosaccharides might be crucial for defining possible and dynamic roles in supporting and suppressing homeostatic cellular processes. The present paper communicates a facile and efficient approach based on glycoblotting method for the characterization of hyaluronan fragments liberated from three different sources of hyaluronan (rooster comb, bovine vitreous humor, and Streptococcus) by in vitro degradation using two typical hyaluronidases of bovine testicular (EC 3.2.1.35) and Streptomyces hyalurolyticus (EC 4.2.2.1). It was demonstrated that glycoblotting method allows for high throughput and quantitative analysis of hyaluronan fragments within a wide dynamic range (1 ~ 1,000 pmole) when 5 µg of hyaluronan digests were applied for this enrichment protocol. Molecular size and distribution of hyaluronan fragments were proved to be influenced strongly by conditions and hyaluronidases employed while source of hyaluronan did not affect the degradation profiles. Strikingly, the present method uncovered the existence of the smallest and unusual hyaluronan degradation fragments such as a disaccharide GlcAß1-3GlcNAc during the digestion by bovine hyaluronidase and a trisaccharide GlcAß1-3GlcNAcß1-4GlcA derivative by Streptomyces hyaluronidase. Bovine testis hyaluronidases afforded hyaluronan tetra- and hexasaccharides as major products. On the other hand, it was demonstrated that Streptomyces hyaluronidase can produce odd number fragments from three to nine sugar residues while even number fragments from four to fourteen sugar residues were major products.

Fentanyl sparing effect of ultrasound-guided proximal radial, ulnar, median, and musculocutaneous nerve (RUMM) block for radial and ulnar fracture repair in dogs: a retrospective case-control study.

This study retrospectively evaluated the fentanyl-sparing effect of ultrasound-guided proximal radial, ulnar, median, and musculocutaneous nerve (RUMM) block for radial and ulnar fracture repair in dogs. Fentanyl was prepared for intraoperative analgesia in dogs, although proximal RUMM block was performed using 0.5% or 0.25% bupivacaine before surgery in the block group. Dogs without a nerve block were assigned to the control group. The fentanyl dose in the block group [0.8 (0-1.9) µg/kg/hr] [median (interquartile range)] was significantly lower than in the control group [8.4 (7.2-10) µg/kg/hr]. Surgery was performed without fentanyl in >50% of the dogs (5/7), using 0.5% bupivacaine. Ultrasound-guided proximal RUMM block can be useful as an intraoperative analgesic for radial and ulnar fracture repair in dogs.

Discovery of Novel NLRP3 Inflammasome Inhibitors Composed of an Oxazole Scaffold Bearing an Acylsulfamide.

The NLRP3 inflammasome plays an important role in the defense mechanism of the innate immune system and has recently attracted much attention as a drug target for various inflammatory disorders. Among the strategies for generating the novel chemotype in current drug discovery, scaffold hopping and bioisosteric replacement are known to be attractive approaches. As the results of our medicinal chemistry campaign, which involved exploration of core motifs using a ring closing approach, a five-membered oxazole-based scaffold was identified, and subsequent implementation of bioisosteric replacement led to discovery of a novel chemical class of NLRP3 inflammasome inhibitor bearing the acylsulfamide group. Further optimization of aniline and sulfamide moieties to improve potency in human whole blood assay led to the identification of the orally bioactive compound 32 in the LPS challenge model. Furthermore, compound 32 attenuated kidney injury in adriamycin-induced glomerulonephritis in mice. These investigations indicated that the NLRP3 inhibitor could be a potential therapeutic agent for glomerulonephritis.

Modular synthesis of phenanthridine derivatives by oxidative cyclization of 2-isocyanobiphenyls with organoboron reagents.

Where HAS you been? A manganese-mediated annulation of 2-isocyanobiaryls with organoboronic acids is developed for the synthesis of a broad range of phenanthridine derivatives. Mechanistic studies indicate that the reaction proceeds by the intramolecular homolytic aromatic substitution (HAS) of an imidoyl radical intermediate.

The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) Prevents Enterocyte Cell Death under Oxygen-Dissolving Cold Conditions through Polyphyletic Antioxidant Actions.

Cold preservation in University of Wisconsin (UW) solution is not enough to maintain the viability of the small intestine, due to the oxidative stress. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual properties to reduce oxidative stress, radical scavenging, and antioxidant protein induction, in other cells. This study was designed to determine whether DHMBA reduces cold preservation injury of enterocytes, and to identify the effector site. Enterocytes were subjected to 48-h cold preservation under atmosphere in UW solution (±DHMBA), and then returned to normal culture to replicate reperfusion of the small intestine after cold preservation. At the end of cold preservation (ECP) and at 1, 3, 6, and 72 h after rewarming (R1h, R3h, R6h, and R72h), we evaluated cell function and the injury mechanism. The results showed that DHMBA protected mitochondrial function mainly during cold preservation, and suppressed cell death after rewarming, as shown by the MTT, ATP, mitochondrial membrane potential, LDH, and lipid peroxidation assays, together with enhanced survival signals (PI3K, Akt, p70S6K) and induction of antioxidant proteins (HO-1, NQO-1, TRX-1). We found that DHMBA mitigates the cold-induced injury of enterocytes by protecting the mitochondria through direct and indirect antioxidative activities.

Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes.

A fluorescence microscopic method for characterizing size, quantity, and oxidation of lipid droplets (LDs) in HepG2 cells was developed. LDs were induced by palmitic (PA), oleic (OA), or linoleic acids (LA) and stained with two fluorescent probes for neutral lipids and lipid peroxides. Each fatty acid increased the number of LDs and oxidized LDs (oxLDs) and the degree of LD oxidation time dependently, as well as increased intracellular triglyceride hydroperoxides. LDs induced by LA without 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) showed the most significant oxidation degree over PA and OA, especially in large LDs (area ≥ 3 µm2, oxLD/LD = 52.3 ± 21.7%). Under this condition, two food-derived antioxidants were evaluated, and both of them significantly improved the LD characteristics. Moreover, chlorogenic acid reduced the quantity of large LDs by 74.0-87.6% in a dose-dependent manner. The proposed method provides a new approach to evaluate the effect of dietary antioxidants on LD characteristics.

Flazin as a Promising Nrf2 Pathway Activator.

Flazin is a ß-carboline-derived alkaloid found in Japanese fermented foods. Here, the potential of flazin as an antioxidant food was studied with particular reference to its effect on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system in human hepatocytes (C3A). Flazin and flazin analogues including the decarboxylated derivative perlolyrine were chemically synthesized and compared with each other and with chlorogenic acid and curcumin. Among these compounds, flazin showed the lowest cytotoxicity (IC50 < 500 µM) and the highest capacity to activate the Keap1-Nrf2 system. It provided the largest (>3-fold of the control) cytoprotection ability against a pro-oxidant, although its radical absorbance capacity was relatively low. Flazin increased the expressions of Nrf2-dependent phase II enzyme genes and their products (NQO1, GSTP, and GSH proteins). The strong cytoprotection ability of flazin associated with low log P (0-3) is shared by sulforaphane and 3,5-dihydroxy-4-methoxybenzyl alcohol, suggesting the potential value of flazin and flazin-rich foods for the prevention of oxidation-related health disorders.

Modification at the 2'-Position of the 4,5-Series of 2-Deoxystreptamine Aminoglycoside Antibiotics To Resist Aminoglycoside Modifying Enzymes and Increase Ribosomal Target Selectivity.

A series of derivatives of the 4,5-disubstituted class of 2-deoxystreptamine aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin was prepared and assayed for (i) their ability to inhibit protein synthesis by bacterial ribosomes and by engineered bacterial ribosomes carrying eukaryotic decoding A sites, (ii) antibacterial activity against wild type Gram negative and positive pathogens, and (iii) overcoming resistance due to the presence of aminoacyl transferases acting at the 2'-position. The presence of five suitably positioned residual basic amino groups was found to be necessary for activity to be retained upon removal or alkylation of the 2'-position amine. As alkylation of the 2'-amino group overcomes the action of resistance determinants acting at that position and in addition results in increased selectivity for the prokaryotic over eukaryotic ribosomes, it constitutes an attractive modification for introduction into next generation aminoglycosides. In the neomycin series, the installation of small (formamide) or basic (glycinamide) amido groups on the 2'-amino group is tolerated.

Examining the effect of regioisomerism on the physico-chemical properties of lysophosphatidylethanolamine-containing liposomes using fluoro probes.

Lysophospholipids (LysoPLs) receive steadily increasing attention in the area of lipid chemistry and biology. However, the physico-chemical properties of individual LysoPL regioisomers have not yet been investigated. Herein, we report the synthesis of fluoro analogues of lysophosphatidylethanolamines (LPEs) and examine the physico-chemical properties of the LPE regioisomers using chemically synthesized fluoro probes.

Absolute quantification of cholesteryl esters using liquid chromatography-tandem mass spectrometry uncovers novel diagnostic potential of urinary sediment.

BACKGROUND: Urine has been utilized as a source of biomarkers in renal disease. However, urinary lipids have not attracted much attention so far. Here we studied urinary cholesteryl ester (CE) and its relevance in renal disease. METHODS: Quantitative analysis of CE molecular species in serum, urinary supernatant, and urinary sediment from patients with renal disease (N=64) and non-renal disease (N=23) was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and deuterated CEs as internal standards. RESULTS: Validation study showed good precision and accuracy of LC-MS/MS. Many CE species were detected in the urinary sediment and supernatant in the renal disease group, whereas only a few CE species were detected in the other group. In the renal disease group, the sum of the concentrations of all CE species showed a significant correlation between the sediment and the supernatant from urinary samples (r=0.876, p<0.001); however, the composition of CEs was significantly different between them. Further, the composition of CEs of the supernatant was similar to that of the serum. CONCLUSIONS: Our LC-MS/MS analysis uncovered a distinct CE profile in urinary sediment from patients with renal disease, suggesting a possible contribution of CEs in urothelial cells to the development of renal disease.

Further studies on cation clock reactions in glycosylation: observation of a configuration specific intramolecular sulfenyl transfer and isolation and characterization of a tricyclic acetal.

The use of the 2-O-(2-trimethylsilylmethallyl) group as intramolecular nucleophile and cation clock reaction in the glucopyranose series depends on the nature of the glycosyl donor. As previously reported, with trichloroacetimidates the anticipated intramolecular Sakurai reaction proceeds efficiently and is an effective clock, whereas with sulfoxides complications arise. The source of these complications is now shown to be an intramolecular sulfenyl transfer reaction between the tethered allylsilane and the activated sulfoxide. These results illustrate how a different unimolecular clock reaction may be required for a given cation when it is generated from different donors in order to avoid side reactions. The synthesis and cyclization of a 2-O-(3-hydroxypropyl) glucopyranosyl sulfoxide leading on activation to the formation of a trans-fused acetal is also described. The formation of this crystallographically-established trans-fused acetal is discussed in terms of the high effective concentration of the intramolecular nucleophile which leads to a high degree of a SN2 character in the displacement of the α-glucosyl triflate or at the level of the corresponding α-CIP. The possible use of such intramolecular alcohols as clock reactions and their limitations is discussed.