Mass spectrometric study of the dissociation of Group XI metal complexes with fatty acids and glycerolipids : Ag2H+ and Cu2H+ ion formation in the presence of a double bond

Results of mass spectrometric studies are reported for the collisional dissociation of Group XI (Cu, Ag, Au) metal ion complexes with fatty acids (palmitic, oleic, linoleic and a-linolenic) and glycerolipids. Remarkably, the formation of M2H+ ions (M = Cu, Ag) is observed as a dissociation product of the ion complexes containing more than one metal cation and only if the lipid in the complex contains a double bond. Ag2H+ is formed as the main dissociation channel for all three of the fatty acids containing double bonds that were investigated while Cu2H+ is formed with one of the fatty acids and, although abundant, is not the dominant dissociation channel. Also. Cu(I) and Ag(I) ion complexes were observed with glycerolipids (including triacylglycerols and glycerophospholipids) containing either saturated or unsaturated fatty acid substituents. Interestingly. Ag2H+ ion is formed in a major fragmentation channel with the lipids that are able to form the complex with two metal cations (triacylglycerols and glycerophosphoglycerols), while lipids containing a fixed positive charge (glycerophospocholines) complex only with a single metal cation. The formation of Ag2H+ ion is a significant dissociation channel from the complex ion Ag-2(L-H)(+) where L = Glycerophospholipid (GP) (18:1/18:1). Cu(I) also forms complexes of two metal cations with glycerophospholipids but these do not produce Cu2H+ upon dissociation. Rather organic fragments, not containing Cu(I), are formed, perhaps due to different interactions of these metal cations with lipids resulting from the much smaller ionic radius of Cu(I) compared to Ag(I) (C).

Influence of cytochrome P-450 inhibitors on the inhalative uptake of methyl chloride and methylene chloride in male B6C3F1 mice

Dichloromethane (DCM) is thought to be metabolized in vivo by two independent pathways: a glutathione (GSH) dependent pathway that yields CO2 and a cytochrome P-450 mediated one that yields both CO and CO2 (Gargas et al 1986). With a physiologically based pharmacokinetic (PB-PK) model, Andersen et al (1987) calculate the quantitative parameters for both metabolic pathways. Using the kinetic parameters thus obtained and the results of two carcinogenicity studies with rodents (Serota et al 1986; NTP 1985), the authors then estimate the tumour risk for humans.

A soluble preparation from developing groundnut seeds (Arachis hypogaea) catalyzes de novo synthesis of long chain fatty acids

A 100,000 x g supernatant fraction prepared from developing groundnut seeds (30-35 days after flowering) catalyzed the synthesis of fatty acids from [l-14C]acetate at a rate of 120nmoles of acetate incorporated per hr per gram fresh weight of tissue. 90% of this incorporated label was associated with fatty acids. The major fatty acids formed were stearic- (77%) and palmitic acids (14%) with 4% of oleic acid. The fatty acid synthetase activity was stable when stored at 0-4 degrees C for at least fifteen days. It is concluded from these results that acetyl-coA carboxylase and all the enzymes of fatty acid synthetase from developing groundnut seeds are soluble.

Frictional response of fatty acids on steel

Self-assembled monolayers of fatty acids were formed on stainless steel by room-temperature solution deposition. The acids are covalently bound to the Surface as carboxylate in a bidentate manner. To explore the effect Of Saturation in the carbon backbone on friction in sliding tribology, we Study the response of saturated stearic acid (SA) and unsaturated linoleic acid (LA) as self-assembled monolayers using lateral force microscopy and nanotribometry and when the molecules are dispersed in hexadecane, using pin-on-disc tribometry. Over a very wide range (10 MPa-2.5 GPa) of contact pressures it is consistently demonstrated that the unsaturated linoleic acid molecules yield friction which is significantly lower than that of the saturated stearic acid. it is argued, using density functional theory predictions and XPS of slid track, that when the molecular backbone of unsaturated fatty acids are tilted and pressed strongly by a probe, in tribological contact, the high charge density of the double bond region of the backbone allows coupling with the steel Substrate. The interaction yields a low friction carboxylate soap film on the substrate. The saturated fatty acid does not show this effect.

Studies in terpenoids. Part 42. Synthesis of 3-(2-hydroxyisopropyl)5,8-dimethyl-1,2-naphthoquinone (emmotin-H)

Emmotin-H, a naturally occurring sesquiterpenoid 1,2-naphthoquinone pigment (1) has been synthesised in a four step sequence starting from the known 5,8-dimethyl-4-oxotetralin-2-carboxylic acid (3a). Selenium dioxide oxidation of its methyl ester (3b) gives 3-methoxycarbonyl-5,8-dimethyl-1,2-naphthoquinone (4) which on reductive acetylation affords the corresponding diacetoxynaphthalene ester (5). Its reaction with excess of methylmagnesium iodide is accompanied by aerial oxidation during work-up and furnishes emmotin-H (1).

Purification of pharmaceuticals and nutraceutical compounds by sub- and supercritical extraction and chromatography

This thesis discusses the use of sub- and supercritical fluids as the medium in extraction and chromatography. Super- and subcritical extraction was used to separate essential oils from herbal plant Angelica archangelica. The effect of extraction parameters was studied and sensory analyses of the extracts were done by an expert panel. The results of the sensory analyses were compared to the analytically determined contents of the extracts. Sub- and supercritical fluid chromatography (SFC) was used to separate and purify high-value pharmaceuticals. Chiral SFC was used to separate the enantiomers of racemic mixtures of pharmaceutical compounds. Very low (cryogenic) temperatures were applied to substantially enhance the separation efficiency of chiral SFC. The thermodynamic aspects affecting the resolving ability of chiral stationary phases are briefly reviewed. The process production rate which is a key factor in industrial chromatography was optimized by empirical multivariate methods. General linear model was used to optimize the separation of omega-3 fatty acid ethyl esters from esterized fish oil by using reversed-phase SFC. Chiral separation of racemic mixtures of guaifenesin and ferulic acid dimer ethyl ester was optimized by using response surface method with three variables per time. It was found that by optimizing four variables (temperature, load, flowate and modifier content) the production rate of the chiral resolution of racemic guaifenesin by cryogenic SFC could be increased severalfold compared to published results of similar application. A novel pressure-compensated design of industrial high pressure chromatographic column was introduced, using the technology developed in building the deep-sea submersibles (Mir 1 and 2). A demonstration SFC plant was built and the immunosuppressant drug cyclosporine A was purified to meet the requirements of US Pharmacopoeia. A smaller semi-pilot size column with similar design was used for cryogenic chiral separation of aromatase inhibitor Finrozole for use in its development phase 2.

Activating Mango Fruit Defence to Anthracnose Disease

The fungus causing anthracnose disease in mango, Colletotrichum gloeosporioides, (C g.), infects immature fruit early in the season, then enters a long latent phase. After harvest, when fruit start to ripen, the latency breaks and the fungus ramifies through the peel and pulp tissues causing black disease lesions. The breaking of pathogen latency in ripening mango fruit has been correlated with decreasing concentrations of the endogenous antifungal resorcinol compounds (Droby et al., 1986). The level of these antifungal resorcinols vary among mango cultivars (Droby et a1 , 1986). Controlling diseases by managing natural resistance of fruit to fungal attack could minimize the use of pesticides, which have become of major public concern on health and environmental grounds. The plant resistance activator benzo(l,2,3)thiadiazole-7-carbothioic acid S-methyl ester (trade name Bion®) has been widely reported as an effective inducer of systemic resistance. For example, Bion® was reported to induce pathogenesis-related proteins (PR proteins) and stimulate plant defence in peas (Dann and Deverall, 2000) and roses (Suo and Leung, 2001). However, until now, there is no information about the role of Bion® in activation of mango (cv. Kensington Pride) fruit resistance to anthracnose disease. The aim of this research is to determine the effect of resistance activators on defence responses of mango fruit to anthracnose disease.

Alamethicin and synthetic peptide fragments as uncouplers of mitochondrial oxidative phosphorylation. Effect of chain length and change

Alamethicin, its derivatives and some synthetic fragments have been shown to be uncouplers of oxidative phosphorylation in rat liver mitochondria. A minimum peptide chain length of 13 residues is necessary for this activity. Peptide esters are more efficient uncouplers than the corresponding peptide acids. Esterification of the Glu(18) γ-COOH group in alamethicin does not diminish uncoupling activity. The structural requirements for uncoupling activity parallel those determined for ionophoretic action in small, unilamellar liposomes. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; Ac, acetyl; CTC, chlortetracycline.

Molecular structure of a cyclic tetrapeptide disulfide. A novel 310 helical conformation with an S-S bridge

The crystal structure of the cyclic peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe has been determined by X-ray diffraction. The peptide crystallizes in the space group P212121, with A = 8.646(1), B = 18.462(2), C = 19.678(3)Å and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4→ 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 310 helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations. Aib, α-aminoisobutyric acid; Z, benzyloxycarbonyl; Boc, t-butyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; NHMe, N-methylamide; Tosyl, p-toluenesulfonyl.

Hydrolysis of sphingomyelin to ceramide with hydrofluoric acid

Sphingomyelin was hydrolyzed to ceramide near quantitatively on treatment with 40% HF at 40°C for 72 hr. The reaction of sphingomyelin with HF is much slower than phosphoglycerides. HF treatment did not alter either the fatty acid composition or the stereochemical configuration of the sphingosine moiety of ceramide formed.

Lipids of some thermophilic fungi

Total lipid content in the thermophilic fungi—Thermoascus aurantiacus, Humicola lanuginosa, Malbranchea pulchella var.sulfurea, andAbsidia ramosa—varied from 5.3 to 19.1% of mycelial dry weight. The neutral and polar lipid fractions accounted for 56.4 to 80.2% and 19.8 to 43.6%, respectively. All the fungi contained monoglycerides, diglycerides, triglycerides, free fatty acids, and sterols in variable amounts. Sterol ester was detected only inA. ramosa. Phosphatide composition was: phosphatidyl choline (15.9–47%), phosphatidyl ethanolamine (23.4–67%), phosphatidyl serine (9.3–17.6%), and phosphatidyl inositol (1.9–11.9%). Diphosphatidyl glycerol occurred in considerable quantity only inH. lanuginosa andM. pulchella var.sulfurea. Phosphatidic acid, detected as a minor component only inM. pulchella var.sulfurea andA. ramosa, does not appear to be a characteristic phosphatide of thermophilic fungi as suggested earlier. The 16∶0, 16∶1, 18∶0, 18∶1, and 18∶2 acids were the main fatty acid components. In addition,A. ramosa contained 18∶3 acid. Total lipids contained an average of 0.93 double bonds per mole of fatty acids, and neutral lipids tend to be more unsaturated than phospholipids.

On the reactivity of carboxyl groups of ribonuclease-A in anhydrous methanol

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens.

1. The biopotencies relative to beta-carotene of several apocarotenoids, such as 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate, were investigated in rats, on a molar basis, by both curative-growth assay and liver-storage tests. 2. In the curative-growth assays, on a molar basis the biopotencies of 8'-, 10'- and 12'-apo-beta-carotenal and methyl 8'-apo-beta-carotenoate were 72, 78, 72 and 53% respectively, whereas on a weight basis the corresponding values were 93, 111, 111 and 63%, with respect to beta-carotene taken as 100%. In terms of yield of vitamin A, these values were much lower in the liver-storage tests. 3. When 8'-apo-beta-carotenal was fed, the unchanged aldehyde together with small amounts of the corresponding alcohol and larger proportions of the acid rapidly appeared in the tissues of both rats and chickens. The 8'-apocarotenol, 8'-apocarotenoic acid and its methyl ester were absorbed unchanged. The free acid disappeared most rapidly from the tissues, but its methyl ester persisted in the tissues longest. 4. On the basis of these observations it is suggested that most of an apocarotenal is oxidized to the corresponding acid, which, in turn, is mostly degraded to retinoic acid, with small proportions of it being attacked by the dioxygenase system giving retinal.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

X-Pro peptides: Synthesis and solution conformation of benzyloxycarbonyl-(Aib-Pro)4methyl ester. Evidence for a novel helical structure

The synthesis of the octapeptide, benzyloxycarbonyl-(-aminoisobutyryl-L-prolyl)4-methyl ester [Z-(Aib-Pro)4-OMe] and an analysis of its solution conformation is reported. The octapeptide is shown to possess three strong intramolecular hydrogen bonds on the basis of studies of the solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. 13C studies are consistent with a structure involving only trans Aib-Pro bonds, while ir experiments support a hydrogen-bonded conformation. The Aib 3, 5, and 7 NH groups are shown to participate in hydrogen bonding. A 310 helical conformation compatible with the spectroscopic data is suggested. The proposed conformation consists of three type III -turns with Aib and Pro at the corners and stabilized by 4 1 intramolecular hydrogen bonds.