Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKQ, which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenyielenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis (2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox(R), a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox(R). Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Liposomes and skin: From drug delivery to model membranes

The early eighties saw the introduction of liposomes as skin drug delivery systems, initially promoted primarily for localised effects with minimal systemic delivery. Subsequently, a novel ultradeformable vesicular system (termed "Transfersomes" by the inventors) was reported for transdermal delivery with an efficiency similar to subcutaneous injection. Further research illustrated that the mechanisms of liposome action depended on the application regime and the vesicle composition and morphology. Ethical, health and supply problems with human skin have encouraged researchers to use skin models. 'IYaditional models involved polymer membranes and animal tissue, but whilst of value for release studies, such models are not always good mimics for the complex human skin barrier, particularly with respect to the stratum corneal intercellular lipid domains. These lipids have a multiply bilayered organization, a composition and organization somewhat similar to liposomes, Consequently researchers have used vesicles as skin model membranes. Early work first employed phospholipid liposomes and tested their interactions with skin penetration enhancers, typically using thermal analysis and spectroscopic analyses. Another approach probed how incorporation of compounds into liposomes led to the loss of entrapped markers, analogous to "fluidization" of stratum corneum lipids on treatment with a penetration enhancer. Subsequently scientists employed liposomes formulated with skin lipids in these types of studies. Following a brief description of the nature of the skin barrier to transdermal drug delivery and the use of liposomes in drug delivery through skin, this article critically reviews the relevance of using different types of vesicles as a model for human skin in permeation enhancement studies, concentrating primarily on liposomes after briefly surveying older models. The validity of different types of liposome is considered and traditional skin models are compared to vesicular model membranes for their precision and accuracy as skin membrane mimics. (c) 2008 Elsevier B.V. All rights reserved.

Can drug-bearing liposomes penetrate intact skin?

Using liposomes to deliver drugs to and through human skin is controversial, as their function varies with type and composition. Thus they may act as drug carriers controlling release of the medicinal agent. Alternatively, they may provide a localized depot in the skin so minimizing systemic effects or can be used for targeting delivery to skin appendages (hair follicles and sweat glands). Liposomes may also enhance transdermal drug delivery, increasing systemic drug concentrations. With such a multiplicity of functions, it is not surprising that mechanisms of liposomal delivery of therapeutic agents to and through the skin are unclear. Accordingly, this article provides an overview of the modes and mechanisms of action of different vesicles as drug delivery vectors in human skin. Our conclusion is that vesicles, depending on the composition and method of preparation, can vary with respect to size, lamellarity, charge, membrane fluidity or elasticity and drug entrapment. This variability allows for multiple functions ranging from local to transdermal effects. Application to dissimilar skins (animal or human) via diverse protocols may reveal different mechanisms of action with possible vesicle skin penetration reaching different depths, from surface assimilation to (rarely) the viable tissue and subsequent systemic absorption.

Drug interaction and location in liposomes: correlation with polar surface areas

An important step in liposome characterization is to determine the location of a drug within the liposome. This work thus investigated the interaction of dipalmitoylphosphatidylcholine liposomes with drugs of varied water solubility, polar surface area (PSA) and partition coefficient using high sensitivity differential scanning calorimetry. Lipophilic estradiol (ES) interacted strongest with the acyl chains of the lipid membrane, followed by the somewhat polar 5-fluorouracil (5-FU). Strongly hydrophilic mannitol (MAN) showed no evidence of interaction but water soluble polymers inulin (IN) and an antisense oligonucleotide (OLG), which have very high PSAs, interacted with the lipid head groups. Accordingly, the drugs could be classified as: hydrophilic ones situated in the aqueous core and which may interact with the head groups; those located at the water-bilayer interface with some degree of penetration into the lipid bilayer; those lipophilic drugs constrained within the bilayer. (c) 2004 Elsevier B.V. All rights reserved.

Interactions of surfactants (edge activators) and skin penetration enhancers with liposomes

Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with ipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (Tm). Surfactants also removed the lipid pre-transition but reduced Tm and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower Tm. The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced Tm and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The Tm and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2 mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.

Plant roots release phospholipid surfactants that modify the physical and chemical properties of soil

Plant root mucilages contain powerful surfactants that will alter the interaction of soil solids with water and ions, and the rates of microbial processes. The lipid composition of maize, lupin and wheat root mucilages was analysed by thin layer chromatography and gas chromatography-mass spectrometry. A commercially available phosphatidylcholine (lecithin), chemically similar to the phospholipid surfactants identified in the mucilages, was then used to evaluate its effects on selected soil properties. The lipids found in the mucilages were principally phosphatidylcholines, composed mainly of saturated fatty acids, in contrast to the lipids extracted from root tissues. In soil at low tension, lecithin reduced the water content at any particular tension by as much as 10 and 50% in soil and acid-washed sand, respectively. Lecithin decreased the amount of phosphate adsorption in soil and increased the phosphate concentration in solution by 10%. The surfactant also reduced net rates of ammonium consumption and nitrate production in soil. These experiments provide the first evidence we are aware of that plant-released surfactants will significantly modify the biophysical environment of the rhizosphere.

Vesicular systems for delivering conventional small organic molecules and larger macromolecules to and through human skin

The history of using vesicular systems for drug delivery to and through skin started nearly three decades ago with a study utilizing phospholipid liposomes to improve skin deposition and reduce systemic effects of triamcinolone acetonide. Subsequently, many researchers evaluated liposomes with respect to skin delivery, with the majority of them recording localized effects and relatively few studies showing transdermal delivery effects. Shortly after this, Transfersomes were developed with claims about their ability to deliver their payload into and through the skin with efficiencies similar to subcutaneous administration. Since these vesicles are ultradeformable, they were thought to penetrate intact skin deep enough to reach the systemic circulation. Their mechanisms of action remain controversial with diverse processes being reported. Parallel to this development, other classes of vesicles were produced with ethanol being included into the vesicles to provide flexibility (as in ethosomes) and vesicles were constructed from surfactants and cholesterol (as in niosomes). Thee ultradeformable vesicles showed variable efficiency in delivering low molecular weight and macromolecular drugs. This article will critically evaluate vesicular systems for dermal and transdermal delivery of drugs considering both their efficacy and potential mechanisms of action.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.

High-affinity small molecule-phospholipid complex formation: Binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction Of X-PA = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.

Determination of the total antioxidant activity of fruits and vegetables by a liposome assay

The effects of mixtures of antioxidants on the oxidation of phospholipids have been investigated in large unilamellar liposomes following initiation by 2,2'-azobis(2-aminopropane) dihydrochloride. The lag phase increased linearly with antioxidant concentration. The lag phases of mixtures containing alpha-tocopherol with ascorbic acid showed synergy between the antioxidants, but mixtures of beta-carotene with cc-tocopherol or ascorbic acid were not synergistic. The liposome system was used to investigate the total antioxidant activity of lipid- and water-soluble extracts from 16 samples of fruits, vegetables, and related food products. The water-soluble extracts caused greater increases in lag phase than the lipid-soluble extracts. The lag phase of liposomes containing the water-soluble extracts from fruits and vegetables increased linearly with the total phenolic concentration, with the continental salad extract having the longest lag phase. The lipid-soluble extract from apples caused the largest increase in lag phase of the lipid-soluble extracts. The lag phases of the lipid-soluble and water-soluble extracts of all fruits and vegetables studied were additive, but no synergy was detected. The lag phase of the liposomes containing both the water-soluble and lipid-soluble extracts varied from 611.5 min for the continental salad extracts to 47.5 min for the cauliflower extracts.

Roseisalinus antarcticus gen. nov., sp nov., a novel aerobic bacteriochlorophyll a-producing alpha-proteobacterium isolated from hypersaline Ekho Lake, Antarctica

A Gram-negative, aerobic to microaerophilic rod was isolated from 10 m depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strain was oxidase- and catalase-positive, metabolized a variety of carboxylic acids and sugars and produced lipase. Cells had an absolute requirement for artificial sea water, which could not be replaced by NaCl. A large in vivo absorption band at 870 nm indicated production of bacteriochlorophyll a. The predominant fatty acids of this organism were 16:0 and 18:1omega7c, with 3-OH 10:0, 16:1omega7c and 18:0 in lower amounts. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. Ubiquinone 10 was produced. The DNA G + C content was 67 mol%. 16S rRNA gene sequence comparisons indicated that the isolate represents a member of the Roseobacter clade within the alpha-Proteobacteria. The organism showed no particular relationship to any members of this clade but clustered on the periphery of the genera Jannaschia, Octadecabacter and 'Marinosulfonomonas' and the species Ruegeria gelatinovorans. Distinct morphological, physiological and genotypic differences to these previously described taxa supported the description of a new genus and a novel species, for which the name Roseisalinus antarcticus gen. nov., sp. nov. is proposed. The type strain is EL-88(T) (= DSM 11466(T) = CECT 7023(T)).

Incorporation of cis-9, trans-11 conjugated linoleic acid and vaccenic acid (trans-11 18 : 1) into plasma and leucocyte lipids in healthy men consuming dairy products naturally enriched in these fatty acids

The present study investigated whether consuming dairy products naturally enriched in cis-9, trans-11 (c9,t11) conjugated linoleic acid (CLA) by modification of cattle feed increases the concentration of this isomer in plasma and cellular lipids in healthy men. The study had a double-blind cross-over design. Subjects aged 34-60 years consumed dairy products available from food retailers for 1 week and then either control (0.17 g c9,t11 CLA/d; 0.31 g trans-vaccenic acid (tVA)/d) or CLA-enriched (1.43 g c9,t11 CLA/d; 4.71 g tVA/d) dairy products for 6 weeks. After 7 weeks washout, this was repeated with the alternate products. c9,t11 CLA concentration in plasma lipids was lower after consuming the control products, which may reflect the two-fold greater c9,t11 CLA content of the commercial products. Consuming the CLA-enriched dairy products increased the c9,t11 CLA concentration in plasma phosphatidylcholine (PC) (38 %; P=0.035), triacylglycerol (TAG) (22 %; P < 0.0001) and cholesteryl esters (205 %; P < 0.0001), and in peripheral blood mononuclear cells (PBMC) (238 %; P < 0.0001), while tVA concentration was greater in plasma PC (65 %; P=0.035), TAG (98 %; P=0.001) and PBMC (84 %; P=0.004). Overall, the present study shows that consumption of naturally enriched dairy products in amounts similar to habitual intakes of these foods increased the c9,t11 CLA content of plasma and cellular lipids.