Studies on steroids. CCXXXXV. Determination of 5 beta-cholestanoic acids in human urine by gas chromatography-mass spectrometry with negative ion chemical ionization detection

A method for the determination of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) in human urine by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Unconjugated, glycine- and taurine-conjugated DHCA and THCA labelled with 18O and 2H were used as internal standards. 5 beta-Cholestanoic acids in urine were extracted with a Sep-Pak C18 cartridge, separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20 and, following alkaline hydrolysis of conjugated forms, derivatization into the pentafluorobenzyl ester-dimethylethylsilyl ethers. Subsequent resolution of each fraction into DHCA and THCA was attained by GC on a cross-linked 5% phenylmethylsilicone fused-silica capillary column where 5 beta-cholestanoic acids were monitored with a characteristic carboxylate anion [M-181]- in the NICI mode using isobutane as a reagent gas. The method was applied to separation and determination of 5 beta-cholestanoic acids in urine from a patient with Zellweger syndrome and from healthy volunteers.     

The antiapoptotic effect of low-dose UVB irradiation in NIH3T3 cells involves caspase inhibitions

UVB irradiation is a well-known apoptosis induction factor. However, we have previously found that low doses of UVB irradiation inhibited apoptosis induced by both serum starvation and lack of extracellular matrix, involving a significant inhibition of caspase-3/7 activation. In this study, we report on the relationship between the UVB-induced anti-apoptotic effect and caspase-3/7 inhibition by reactive oxygen species (ROS). The UVB-induced antiapoptotic effect was partially prevented by an antioxidant agent, N-acetylcysteine. A ROS-generating agent, menadione and a pro-oxidant agent, H2O2 also showed an effect that was similar to the UVB-induced antiapoptotic effect, indicating that ROS contributed to the antiapoptotic effect. UVB irradiation significantly suppressed caspase-3/7 activation, which was caused by the inhibition of proteolysis and not by the inhibition of enzymatic activity itself. The prevention of proteolysis was also confirmed by both the following results: one is the inhibition of in vitro caspase-3/7 and -9 activation in cell lysates exposed to UVB in the presence of cytochrome c and dATP, which was caused by the production of ROS, and the other is the inhibition of in vitro caspase-3/7 activation in the presence of active caspase-9. These results showed that the inhibition of the caspase cascade downstream mitochondria by ROS production, leading to a significant inhibition of caspase-3/7 activation, was one of the causes of the antiapoptotic effect by small doses of UVB irradiation.     

Assembly of Carbohydrates on a Nickel(II) Center by Utilizing N-Glycosidic Bond Formation with Tris(2-aminoethyl)amine (tren). Syntheses and Characterization of [Ni{N-(aldosyl)-tren}(H(2)O)](2+), [Ni{N,N'-bis(aldosyl)-tren}](2+) and [Ni{N,N',N"-tris(aldosyl)-tren}](2+)

Reactions of [Ni(tren)(H(2)O)(2)]X(2) (tren = tris(2-aminoethyl)amine; X = Cl (1a), Br (1b); X(2) = SO(4) (1c)) with mannose-type aldoses, having a 2,3-cis configuration (D-mannose and L-rhamnose), afforded {bis(N-aldosyl-2-aminoethyl)(2-aminoethyl)amine}nickel(II) complexes, [Ni(N,N'-(aldosyl)(2)-tren)]X(2) (aldosyl = D-mannosyl, X = Cl (2a), Br (2b), X(2) = SO(4) (2c); aldosyl = L-rhamnosyl, X(2) = SO(4) (3c)). The structure of 1c was confirmed by X-ray crystallography to be a mononuclear [Ni(II)N(4)O(2)] complex with the tren acting as a tetradentate ligand (1c.2H(2)O: orthorhombic, Pbca, a = 15.988(2) ?, b = 18.826(4) ?, c = 10.359(4) ?, V = 3118 ?(3), Z = 8, R = 0.047, and R(w) = 0.042). Complexes 2a,c and 3c were characterized by X-ray analyses to have a mononuclear octahedral Ni(II) structure ligated by a hexadentate N-glycoside ligand, bis(N-aldosyl-2-aminoethyl)(2-aminoethyl)amine (2a.CH(3)OH: orthorhombic, P2(1)2(1)2(1), a = 16.005(3) ?, b = 20.095(4) ?, c = 8.361(1) ?, V = 2689 ?(3), Z = 4, R = 0.040, and R(w) = 0.027. 2c.3CH(3)OH: orthorhombic, P2(1)2(1)2(1), a = 14.93(2) ?, b = 21.823(8) ?, c = 9.746(2) ?, V = 3176 ?(3), Z = 4, R = 0.075, and R(w) = 0.080. 3c.3CH(3)OH: orthorhombic, P2(1)2(1)2(1), a = 14.560(4) ?, b = 21.694(5) ?, c = 9.786(2) ?, V = 3091 ?(3), Z = 4, R = 0.072, and R(w) = 0.079). The sugar part of the complex involves novel intramolecular sugar-sugar hydrogen bondings around the metal center. The similar reaction with D-glucose, D-glucosamine, and D-galactosamine, having a 2,3-trans configuration, resulted in the formation of a mono(sugar) complex, [Ni(N-(aldosyl)-tren)(H(2)O)(2)]Cl(2) (aldosyl = D-glucosyl (4b), 2-amino-2-deoxy-D-glucosyl (5a), and 2-amino-2-deoxy-D-galactosyl (5b)), instead of a bis(sugar) complex. The hydrogen bondings between the sugar moieties as observed in 2 and 3 should be responsible for the assembly of two sugar molecules on the metal center. Reactions of tris(N-aldosyl-2-aminoethyl)amine with nickel(II) salts gave the tris(sugar) complexes, [Ni(N,N',N"-(aldosyl)(3)-tren)]X(2) (aldosyl = D-mannosyl, X = Cl (6a), Br (6b); L-rhamnosyl, X = Cl (7a), Br (7b); D-glucosyl, X = Cl (9); maltosyl, X = Br (10); and melibiosyl, X = Br (11)), which were assumed to have a shuttle-type C(3) symmetrical structure with Delta helical configuration for D-type aldoses on the basis of circular dichroism and (13)C NMR spectra. When tris(N-rhamnosyl)-tren was reacted with NiSO(4).6H(2)O at low temperature, a labile neutral complex, [Ni(N,N',N"-(L-rhamnosyl)(3)-tren)(SO(4))] (8), was successfully isolated and characterized by X-ray crystallography, in which three sugar moieties are anchored only at the N atom of the C-1 position (8.3CH(3)OH.H(2)O: orthorhombic, P2(1)2(1)2(1), a = 16.035(4) ?, b = 16.670(7) ?, c = 15.38(1) ?, V = 4111 ?(3), Z = 4, R = 0.084, and R(w) = 0.068). Complex 8 could be regarded as an intermediate species toward the C(3) symmetrical tris(sugar) complexes 7, and in fact, it was readily transformed to 7b by an action of BaBr(2).     

Selectivity enhancement of diastereomer separation in RPLC using crystalline-organic phase-bonded silica

Summary Poly(octadecyl acrylate) exists in a crystalline state at room temperature. This crystalline state was formed even after being grafted onto silica through the terminal group of the polymer. When this polymer-grafted silica (Sil-ODA_n) was used as a reversed phase liquid chromatography packing material, better selectivity for diastereomerized phenylethylamines derivatized by (S)-(−)-(2,3-naphthalenedicarboximidyl)propionyl fluoride was observed at room temperature than was observed for simple octadecylated silica (ODS). On the other hand, increasing temperature reduced the selectivity to be close to that of ODS. Additional experiments showed the better selectivity derived from Sil-ODA_n was due to both the highly-oriented structure of the polymer and the carbonyl-π interaction with the diastereomer. A theoretical investigation of this carbonyl-π interaction is also described briefly.     

Metal-induced supramolecualr chirality in an optically active polythiophene aggregate

Chiral polythiophenes (PTs), in sharp contrast to other optically active polymers, exhibit optical activity in the pi-pi* transition region which is derived from the chirality of the main chain when they self-assemble to form a supramolecular pi-stacked aggregate with intermolecular interactions in a poor solvent or in a film. We now report that the regioregular, optically active PT poly[(R)-3-[4-(4-ethyl-2-oxazolin-2-yl)phenyl]thiophene] (poly-1) exhibits unique split-type induced circular dichroism (ICD) in the pi-pi* transition region of the main chain upon complexation with various metal salts such as trifluoromethanesulfonates of copper(I), copper(II), silver(I), and zinc(II), and iron(II) perchlorate in chloroform, which is a good solvent for poly-1. The appearance of ICD and slight changes in the UV/Vis spectra (no color change), except for the zinc salts, indicated that the chirality may not be induced by chiral pi-stacked aggregates of poly-1, but by the chirality of the main chain, for example, a predominantly one-handed helical structure induced by intermolecular coordination of the oxazoline groups to metal ions. The sign of the Cotton effect depends on the metal salt; most metal salts induced ICDs with similar Cotton-effect patterns, while zinc salts caused an inversion of the signs of the Cotton effect of poly-1 accompanied by a gradual red shift in the absorption of up to 125 nm. The changes in the conformation and the size of the poly-1 aggregates induced by different metal salts were also investigated by (1)H NMR titrations, static light scattering (SLS), atomic force microscopy (AFM), and membrane filtration. On the basis of these results, we propose a possible model for the chiral supramolecular aggregates of poly-1 with metal salts.     

Total synthesis of slow reacting substances (SRS's): 6-epi-leukotriene C and 6-epi-leukotriene D

6-$\text{Epi}$-leukotrienes C and D (3 and 4) have been synthesized unambiguously via the 5($\text{S}$), 6($\text{R}$)-epoxide (5,6-$\text{cis}$) which is isomeric with leukotriene A. These 6-$\text{epi}$-leukotrienes are less active (especially 4) than leukotrienes C and D (1 and 2) and have not been found in substantial quantity in natural SRS sources.     

Mechanism for the inhibition of the acid degradation of ampicillin by 2-hydroxypropyl- β -cyclodextrin

The formation of inclusion complexes between amoxicillin (AMPC) and 2-hydroxypropyl-β-cyclodextrin (HPCD) was investigated by isothermal microcalorimetry and molecular dynamics simulation to evaluate the inhibitory effects on the degradation of AMPC in aqueous solutions at various pH. The process depended significantly on the ionic species of AMPC in the solution. In a strong acid solution, cationic AMPC and HPCD formed two different types of inclusion complexes with a 1:1 stoichiometry: the first-type had a high association constant K ₁ of 4.0-8.0·10³ M^-1 and included the penam ring of AMPC in the HPCD cavity (Mode I), while the second-type with a K ₂ of 1.0·10³ M^-1 contained the phenyl group of AMPC (Mode II). Furthermore, a complex with a 1:2 (AMPC:HPCD) stoichiometry was realized in a two-step reaction and was characterized by a smaller K _1:2of 4.0·10² M^-1 and larger negative enthalpy and entropy changes than the complexes with a 1:1 stoichiometry. Since the β-lactam ring of AMPC could be protected by inclusion with HPCD in the 1:2 complex and Mode I of 1:1 complexes, the degradation of AMPC in the presence of HPCD was approximately four times slower than in its absence at pH 1.2 and 37°C. In weak acid and neutral solutions, zwitterionic AMPC and HPCD formed only one type of inclusion complex with a 1:1 stoichiometry, where the phenyl group was included (Mode II). AMPC was very stable in these solutions (t _1/2=226 h at pH=6.0) and there is little significant difference in the degradation rate between complexed AMPC and uncomplexed AMPC. Thus, the results indicated that the inclusion complex of AMPC with HPCD, effectively increasing the stability of AMPC in a strong acidic solution like that the stomach, would be useful for eradicating Helicobacter pylori infection and as a drug delivery system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid and simultaneous analysis of dichlorvos, malathion, carbaryl, and 2,4-dichlorophenoxy acetic acid in citrus fruit by flow-injection ion spray ionization tandem mass spectrometry

A simple method for the rapid and simultaneous analysis of dichlorvos (DDVP), malathion, carbaryl, and 2,4-dichlorophenoxy acetic acid (2,4-D) in citrus fruit, which uses flow-injection ion spray ionization tandem mass spectrometry, has been developed for the first time. The method involves the combined use of stable isotopically labeled internal standards (DDVP-d₆, malathion-d₁₀, carbaryl-d₇, and 2,4-D-d₅) and a multiple reaction monitoring technique. The average recoveries for the pesticides at the same concentrations as their tolerance levels (DDVP: 0.1-0.2 μg g⁻¹; malathion: 0.5-4.0 μg g⁻¹; carbaryl: 1.0 μg g⁻¹; 2,4-D: 1.0-2.0 μg g⁻¹) ranged from 90 to 119% with the relative standard deviation (R.S.D.) ranging from 1.0 to 13.1% (n = 5). Analysis time, including sample preparation and determination, was only 15 min. The present method is effective for screening DDVP, malathion, carbaryl, and 2,4-D in citrus fruit.     

Purification of Food Color Red No. 106 (acid red) using pH-zone-refining counter-current chromatography

pH-Zone-refining counter-current chromatography was successfully applied to the separation of the main components of Food Color Red No. 106 (R-106, acid red, Color Index No. 45100). A 300-mg quantity of sample was separated using the following two-phase solvent system: n-butanol-water, 40 mM sulfuric acid in organic stationary phase and 30 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by high-performance liquid chromatography and fast atom bombardment mass spectrometry. The separation yielded 261.9 mg of main component of acid red with purity of 99.9%.     

Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes

Simultaneous enantioseparations of 15 racemic aromatic amino acids and L-mimosine for their chiral discrimination were achieved by neutral selector-modified capillary electrophoresis (CE) and by charged selector-modified CE. Among the diverse cyclodextrins (CDs) examined, hydroxypropyl (HP)-alpha-CD as the neutral selector and highly sulfated (HS)-gamma-CD as the charged selector provided best chiral environments of different enantioselectivities. Fairly good enantiomeric resolutions were achieved with the HP-alpha-CD mode except for racemic 6-hydroxy-3,4-dihydroxyphenylalanine, threo-3,4-dihydroxyphenylserine and homophenylalanine while high-resolution separations of all the enantiomeric pairs were achieved in the HS-gamma-CD mode except that L-mimosine was not detected and a partial resolution (0.6) for threo-3,4-dihydroxyphenylserine enantiomers. Relative migration times to that of internal standard under the respective optimum conditions were characteristic of each enantiomer with good precision (% RSD: 0.7-3.8), thereby enabling to cross-check the chemical identification of aromatic amino acids and also their chiralities. The method linearity was found to be adequate (r> 0.99) for the chiral assay of the aromatic amino acids investigated. When applied to extracts of three plant seeds, nonprotein amino acids such as L-mimosine (42 nug/g) from Mimosa pudica Linné, and L-3,4-dihydroxyphenylalanine (268 nug/g) from Vicia faba were positively detected along with L-tryptophan, L-phenylalanine and L-tyrosine.