Gas-phase rate coefficients for the reactions of NO3, OH and O-3 with alpha,beta-unsaturated esters and ketones: structure-activity relations (SARs)

Gas-phase rate coefficients for the atmospherically important reactions of NO3, OH and O-3 are predicted for 55 alpha,beta-unsaturated esters and ketones. The rate coefficients were calculated using a correlation described previously [Pfrang, C., King, M.D., C. E. Canosa-Mas, C.E., Wayne, R.P., 2006. Atmospheric Environment 40, 1170-1179]. These rate coefficients were used to extend structure-activity relations for predicting the rate coefficients for the reactions of NO3, OH or O-3 with alkenes to include alpha,beta-unsaturated esters and ketones. Conjugation of an alkene with an alpha,beta-keto or alpha,beta-ester group will reduce the value of a rate coefficient by a factor of similar to 110, similar to 2.5 and similar to 12 for reaction with NO3, OH or O-3, respectively. The actual identity of the alkyl group, R, in -C(O)R or -C(O)OR has only a small influence. An assessment of the reliability of the SAR is given that demonstrates that it is useful for reactions involving NO3 and OH, but less valuable for those of O-3 or peroxy nitrate esters. (c) 2006 Elsevier Ltd. All rights reserved.

First efficient and general copper-catalyzed [2,3]-rearrangement of tetrahydropyridinium ylids

The factors affecting the copper-catalyzed rearrangement of ammonium ylids derived from tetrahydropyriclines and diazoesters; have been examined,and the first examples of high-yielding metal-catalyzed [2,3]-sigmatropic rearrangements of a wide range of such ylids are reported. The nature of the alpha-substituent in the diazo component of the reaction has a dramatic effect upon the yields of the reaction, with electron-withdrawing substituents enhancing the yield of the reaction.

Carbanucleosides: synthesis of both enantiomers of 2-(6-chloro-purin-9-yl)-3,5-bishydroxymethyl cyclopentanol from D-glucose

The key intermediate 1,2:5,6-di-O-isopropylidene-3-deoxy-3 beta-allyl-alpha-D-glucofuranose (8) could be conveniently prepared through radical induced allyl substitution at C-3 of appropriate 1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose derivatives (7a,b) and used to synthesize enantiomeric bishydroxymethyl aminocyclopentanols 13 and 19 by the application of a 1,3-dipolar nitrone cycloaddition reaction involving the C-5 or C-1 aldehyde functionality. The products were subsequently transformed into carbanucleoside enantiomers 15 and 21. The diastercomeric isoxazolidinocyclopentane derivative 20 was similarly converted to carbanucleoside 22. (c) 2006 Elsevier Ltd. All rights reserved.

Noncompetitive plasma biokinetics of deuterium-labeled natural and synthetic alpha-tocopherol in healthy men with an apoE4 genotype

Previous studies comparing the biokinetics of deuterated natural (RRR) and synthetic (all-rac) &alpha;-tocopherol (vitamin E) used a simultaneous ingestion or competitive uptake approach and reported relative bioavailability ratios close to 2:1, higher than the accepted biopotency ratio of 1.36:1. The aim of the current study was to compare the biokinetics of deuterated natural and synthetic vitamin E using a noncompetitive uptake model both before and after vitamin E supplementation in a distinct population. Healthy men (n = 10) carrying the apolipoprotein (apo)E4 genotype completed a randomized crossover study, comprised of two 4-wk treatments with 400 mg/d (RRR-&alpha;-tocopheryl and all-rac-&alpha;-tocopheryl acetate) with a 12-wk washout period between treatments. Before and after each treatment period, the subjects consumed a capsule containing 150 mg deuterated &alpha;-tocopheryl acetate in either the PRR or all-rac form depending on their treatment regimen. Blood was obtained up to 48 h after ingestion, and tocopherols analyzed by LC/MS. After deuterated all-rac administration, plasma deuterated tocopherol maximum concentrations and area under the concentration vs. time curves (AUC) were lower than those following RRR administration. The RRR:all-rac ratios determined from the deuterated biokinetic profiles (maximum concentration; C-max) and AUCs were 1.35:1 &PLUSMN; 0.17 and 1.33:1 &PLUSMN; 0.18, respectively. The 4-wk supplementation with either PRR or all-rac significantly increased plasma a-tocopherol concentrations (P < 0.001), but decreased the plasma response to newly absorbed deuterated RRR or all-rac &alpha;-tocopherol. Using a noncompetitive uptake approach, the relative bioavailability of natural to synthetic vitamin E in apoE4 males was close to the currently accepted biopotency ratio of 1.36:1.

Cigarette smokers differ in their handling of natural (RRR) and synthetic (all rac) alpha-tocopherol: a biokinetic study in apoE4 male subjects

We have compared the biokinetics of deuterated natural (RPR) and synthetic (all rac) alpha-tocopherol in male apoE4-carrying smokers and nonsmokers. In a randomized, crossover study subjects underwent two 4-week treatments (400 mg/day) with undeuterated RRR- and all rac-alpha-tocopheryl acetate around a 12-week washout. Before and after each supplementation period subjects underwent a biokinetic protocol (48 h) with 150 mg deuterated RRR- or all rac-alpha-tocopheryl acetate. During the biokinetic protocols, the elimination of endogenous plasma alpha-tocopherol was significantly faster in smokers (P < 0.05). However, smokers had a lower uptake of deuterated RRR than nonsmokers, but there was no difference in uptake of deuterated all rac. The supplementation regimes significantly raised plasma alpha-tocopherol (P < 0.001) with no differences in response between smokers and nonsmokers or between alpha-tocopherol forms. Smokers had significantly lower excretion of alpha-carboxyethyl-hydroxychroman than nonsmokers following supplementation (P < 0.05). Nonsmokers excreted more alpha-carboxyethyl-hydroxychroman following RRR than all rac; however, smokers did not differ in excretion between forms. At baseline, smokers had significantly lower ascorbate (P < 0.01) and higher F(2-)isoprostarres (P < 0.05). F-2-isoprostanes in smokers remained unchanged during the study, but increased in nonsmokers following alpha-tocopherol supplementation. These data suggest that apoE4-carrying smokers and nonsmokers differ in their handling of natural and synthetic alpha-tocopherol. (c) 2006 Elsevier Inc. All rights reserved.

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha-D-mannopyranosyl-(1-->2)-D-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha-D-mannopyranosyl-(1-->3)-D-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as 'decoys' of receptors of type-1 fimbriae carried by enterobacteria.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Cigarette smokers have decreased lymphocyte and platelet alpha-tocopherol levels and increased excretion of the gamma-tocopherol metabolite gamma-carboxyethyl-hydroxychroman (gamma-CEHC)

Cigarette smoking is associated with increased oxidative stress and increased risk of degenerative disease. As the major lipophilic antioxidant, requirements for vitamin E may be higher in smokers due to increased utilisation. In this observational study we have compared vitamin E status in smokers and non-smokers using a holistic approach by measuring plasma, erythrocyte, lymphocyte and platelet alpha- and gamma-tocopherol, as well as the specific urinary vitamin E metabolites alpha- and gamma-carboxyethylhydroxychroman (CEHC). Fifteen smokers (average age 27 years, smoking time 7.5 years) and non-smokers of comparable age, gender and body mass index (BMI) were recruited. Subjects completed a 7-day food diary and on the final day they provided a 24 h urine collection and a 20 ml blood sample for measurement of urinary vitamin E metabolites and total vitamin E in blood components, respectively. No significant differences were found between plasma and erythrocyte alpha- and gamma-tocopherol in smokers and non-smokers. However, smokers had significantly lower ce-tocopherol (mean +/-SD, 1.34+/-0.31 mumol/g protein compared with 1.94+/-0.54, P = 0.001) and gamma-tocopherol (0.19 +/- 0.04 mumol/g protein compared with 0.26 +/- 0.08, P = 0.026) levels in their lymphocytes, as well as significantly lower (alpha-tocopherol levels in platelets (1.09 +/- 0.49 mumol/g protein compared with 1.60 +/- 0.55, P = 0.014; gamma-tocopherol levels were similar). Interestingly smokers also had significantly higher excretion of the urinary gamma-tocopherol metabolite, gamma-CEHC (0.49 +/- 0.25 mg/g creatinine compared with 0.32 +/- 0.16, P = 0.036) compared to non-smokers, while their (alpha-CEHC (metabolite of a-tocopherol) levels were similar. There was no significant difference between plasma ascorbate, urate and F-2-isoprostane levels. Therefore in this population of cigarette smokers (mean age 27 years, mean smoking duration 7.5 years), alterations to vitamin E status can be observed even without the more characteristic changes to ascorbate and F-2-isoprostanes. We suggest that the measurement of lymphocyte and platelet vitamin E may represent a valuable biomarker of vitamin E status in relation to oxidative stress conditions.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

A novel alpha-galactosidase from I'ifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of a parts per thousand 243 kDa and a subunit size of a parts per thousand 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (a parts per thousand 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) a parts per thousand yen3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

Effect of altered dietary n-3 fatty acid intake upon plasma lipid fatty acid composition, conversion of [C-13]alpha-linolenic acid to longer-chain fatty acids and partitioning towards beta-oxidation in older men

The effect of increased dietary intakes of alpha-linolenic acid (ALNA) or eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for 2 months upon plasma lipid composition and capacity for conversion of ALNA to longer-chain metabolites was investigated in healthy men (52 (SD 12) years). After a 4-week baseline period when the subjects substituted a control spread, a test meal containing [U-C-13]ALNA (700 mg) was consumed to measure conversion to EPA, docosapentaenoic acid (DPA) and DHA over 48 h. Subjects were then randomised to one of three groups for 8 weeks before repeating the tracer study: (1) continued on same intake (control, n 5); (2) increased ALNA intake (10 g/d, n 4); (3) increased EPA+DHA intake (1.5 g/d, n 5). At baseline, apparent fractional conversion of labelled ALNA was: EPA 2.80, DPA 1.20 and DRA 0.04%. After 8 weeks on the control diet, plasma lipid composition and [C-13]ALNA conversion remained unchanged compared with baseline. The high-ALNA diet resulted in raised plasma triacylglycerol-EPA and -DPA concentrations and phosphatidylcholine-EPA concentration, whilst [C-13]ALNA conversion was similar to baseline. The high-(EPA+DHA) diet raised plasma phosphatidylcholine-EPA and -DHA concentrations, decreased [C-13]ALNA conversion to EPA (2-fold) and DPA (4-fold), whilst [C-13]ALNA conversion to DHA was unchanged. The dietary interventions did not alter partitioning of ALNA towards beta-oxidation. The present results indicate ALNA conversion was down-regulated by increased product (EPA+DHA) availability, but was not up-regulated by increased substrate (ALNA) consumption. This suggests regulation of ALNA conversion may limit the influence of variations in dietary n-3 fatty acid intake on plasma lipid compositions.

Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants

Objective: Certain milk factors may promote the growth of a host-friendly gastrointestinal microbiota, for example, one that is predominated by bifidobacteria, a perceived healthpromoting genus. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts who are believed to have a more diverse microbiota, which is similar to that of adults. The effects of formulas supplemented with 2 such ingredients from bovine milk, a-lactalbumin (alpha-lac) and casein glycomacropeptide (GMP), on gut flora were investigated in this study. Patients and Methods: Six-week-old (4-8 wk), healthy term infants were randomised to a standard infant formula or 1 of 2 test formulae enriched in alpha-Jac with higher or lower GMP until 6 months. Faecal bacteriology was determined by the culture-independent procedure fluorescence in situ hybridisation. Results: There was a large fluctuation of bacterial counts within groups with no statistically significant differences between groups. Although all groups showed a. predominance of bifidobacteria, breast-fed infants had a small temporary increase in counts. Other bacterial levels varied in formula-fed groups, which overall showed an adult-like faecal microflora. Conclusions: It can be speculated that a prebiotic effect for alpha-lac and GMP is achieved only with low starting populations of beneficial microbiota (eg, infants not initially breast-fed.

A two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium

Aims: Certain milk factors may promote the growth of a gastrointestinal microflora predominated by bifidobacteria and may aid in overcoming enteric infections. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Methods and Results: Infant faecal specimens were used to ferment formulae supplemented with glycomacropeptide (GMP) and alpha-lactalbumin (alpha-la) in a two-stage compound continuous culture model. At steady state, all fermenter vessels were inoculated with 5 ml of 0.1 M phosphate-buffered saline (pH 7.2) containing 10(8) CFU ml(-1) of either enteropathogenic Escherichia coli 2348/69 (O127:H6) or Salmonella serotype Typhimurium (DSMZ 5569). Bacteriology was determined by independent fluorescence in situ hybridization. Vessels that contained breast milk (BM), as well as alpha-la and GMP supplemented formula had stable total counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and E. coli decreased significantly in all three groups prior to pathogen addition. Escherichia coli counts decreased in vessels containing BM and alpha-la while Salmonella decreased significantly in all vessels containing BM, alpha-la and GMP. Acetate was the predominant acid. Significance and Impact of the Study: Supplementation of infant formulae with appropriate milk proteins may be useful in mimicking the beneficial bacteriological effects of breast milk.

Effect of apoE genotype and dietary quercetin on blood lipids and TNF-alpha levels in apoE3 and apoE4 targeted gene replacement mice

The aim of this study was to determine the effect of dietary quercetin supplementation on blood lipids and TNF-alpha levels according to the apoE genotype in apoE3 and apoE4 targeted gene replacement mice. In a two-factorial design female apoE3 and apoE4 mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Feeding the quercetin-supplemented diets significantly increased plasma levels of quercetin and isorhamnetin both in apoE3 and apoE4 mice. There was no significant effect of apoE genotype on plasma quercetin levels. ApoE3 and apoE4 transgenic mice exhibited similar plasma levels of apoE and cholesterol which were not significantly affected by dietary quercetin supplementation. In mice receiving the basal diet without quercetin supplementation, levels of TNF-alpha in whole blood stimulated ex vivo with lipopolysaccharide were higher in apoE3 as compared to apoE4 transgenic mice. Dietary quercetin significantly lowered levels of TNF-alpha by 44% in apoE3 mice relative to apoE3 mice receiving the unsupplemented diets. In apoE4 mice a moderate (20%) but not significant decrease in TNF-alpha levels in response to the quercetin supplementation was evident. Following quercetin supplementation TNF-alpha levels were similar between apoE3 and apoE4 transgenic mice. Current findings indicate that apoE3 mice are more responsive to the TNF-alpha lowering properties of dietary quercetin supplementation as compared to apoE4 animals.

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillits phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5. a K-m of 14 mM with alpha-D-Manp-(1 -> 6)-D-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1 -> 6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1 -> 3)-D-Manp 10 times faster than alpha-D-Manp-(1 -> 6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1 -> 2)-D-Manp. The activity towards (1 -> 3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1 -> 6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities. (c) 2005 Elsevier Ltd. All rights reserved.