Anti-inflammatory action of a phosphatidylcholine, phosphatidylethanolamine and N-acylphosphatidylethanolamine-enriched diet in carrageenan-induced pleurisy.

BACKGROUND/AIMS: Phosphatidylcholine (PC)-derived choline exhibits anti-inflammatory properties in stress conditions. Phosphatidylethanolamine (PE) and N-acylphosphatidylethanolamines (NAPEs) are endogenous bioactive phospholipids linked to the PC and endocannabinoid metabolisms. We hypothesized that an increased dietary input of PC, PE and NAPE may interfere with leukocyte reactions and thus decreases the inflammatory activation. METHODS: CFLP mice were fed with a control diet or with a diet supplemented with 1% PC, 0.4% PE and 0.1% NAPE for 7 days before the induction of pleurisy with carrageenan. Pleural leukocyte migration, pulmonary mast cell degranulation (Alcian blue-safranin O staining), and the activities of inducible nitric oxide synthase, xanthine oxidoreductase and myeloperoxidase were determined in lung tissue biopsies. RESULTS: The carrageenan-induced inflammatory response was characterized by pulmonary leukocyte infiltration, mast cell degranulation and significantly increased inducible nitric oxide synthase and xanthine oxidoreductase activities (by 82 and 60%, respectively). Treatment of mice with acetylsalicylic acid or with dietary PC + PE + NAPE supplementation significantly decreased the leukocyte reaction, and suppressed the activity of the pulmonary proinflammatory enzymes. CONCLUSION: This study confirms a potential for dietary PC + PE + NAPE supplementation to influence events crucial for the remission of acute inflammation. PC + PE + NAPE administration could possibly be a novel preventive or pharmacotherapeutic option in inflammatory pathologies.

Endogenous phospholipid metabolite containing topical product inhibits ultraviolet light-induced inflammation and DNA damage in human skin.

BACKGROUND: N-palmitoylethanolamine (PEA) and organic osmolytes are endogenous components of the human epidermis and are generated from phospholipids in the stratum granulosum. PEA has been shown to exert potent antioxidant and anti-inflammatory activities. The endogenous organic osmolytes such as betaine and sarcosine control skin humidity, but have also been shown to inhibit ultraviolet (UV) light-induced oxidative stress in keratinocytes. OBJECTIVES: To investigate the effect of a PEA- and organic osmolyte-containing topical product (Physiogel AI) on the development of UV light-induced erythema, thymine dimer formation and p53 tumor suppressor gene activation, as well as intercellular adhesion molecule 1 (ICAM-1) and Ki67 expression in normal human skin. METHODS: The UV-induced erythema was measured by a spectrofluorometric method. Thymine dimers, p53, ICAM-1 and Ki67 were detected in skin biopsies using immunohistochemistry. RESULTS: Physiogel AI cream significantly inhibited the development of UV light-induced erythema and thymine dimer formation in normal human skin, but did not alter the number of Ki67+ proliferating keratinocytes and the expression of p53 and ICAM-1. CONCLUSIONS: Our results suggest that PEA and organic osmolytes might represent a new generation of compounds which suppress UV-induced photodamage.

Betaine-palmitate reduces acetylsalicylic acid-induced gastric damage in rats.

BACKGROUND: Acetylsalicylic acid (ASA)-induced gastric injury is reduced when ASA is administered along with phosphatidylcholine. The hydrolysis of endogenous phosphatidylcholine leads to the production of betaine, which may participate in the maintenance of cellular homeostasis. The present aims were to investigate the effects of exogenous betaine and its palmitic acid complex (betaine-palmitate) in the protection of the gastric mucosa in ASA-induced subacute damage. METHODS: Repeated doses of ASA were given intragastrically to male Wistar rats. Control rats were given vehicle only, while treated animals were challenged with ASA or with ASA along with betaine, palmitic acid or betaine-palmitate. The gastric mucosa was examined after 3 days and the nature of any microscopic mucosal injury was assessed by histology. The extent of macroscopic damage, changes in permeability (assessed by Evans blue method) and tissue ATP concentrations were determined in separate series. RESULTS: ASA induced a significant fall in the ATP content of the mucosa, which was not affected by the other drugs used in the study. However, the ASA-induced mucosal permeability increase could be completely reversed by betaine-palmitate supplementation. The extent of severity of the macroscopic and microscopic lesions was 33% and 2.45, respectively, for ASA, as compared with 15% and 2.2 for betaine, 14% and 1.9 for palmitic acid and 3% and 1.4 for betaine-palmitate. CONCLUSIONS: Betaine-palmitate affords a significant protective effect against ASA-induced injury, without influencing the ATP synthesis, and this suggests that the defence is due to its ability to prevent secondary damage.

Electrophilic methyl groups present in the diet ameliorate pathological states induced by reductive and oxidative stress: a hypothesis.

Reductive stress, characterised by an increased NADH:NAD+ ratio, may be as common and as important a consequence of redox imbalance as oxidative stress. It may also be an important predisposing cause of the generation of reactive oxygen species. Considerable experimental and indirect clinical evidence suggests that protection against reductive stress depends on biomolecules with electrophilic methyl groups (EMG) such as S-adenosylmethionine, betaine, carnitine and phosphatidylcholine. Pathological processes leading to reductive stress and their relief by such protective agents is reviewed and the proposed molecular mechanism is outlined. These and other EMG-containing biomolecules are part of the daily diet and may represent an important control system for redox balance.

Disturbances of sperm flagella due to failure of epididymal maturation and their possible relationship to phospholipids.

A recent classification of alterations of midpiece and flagellum describes the phenomenon of abnormal staining behaviour of human sperm flagella during Papanicolaou and Shorr staining. In these techniques, human sperm tails normally stain red; however, if epididymal function is disturbed, the flagella appear bluish, while they are devoid of other recognizable defects. Such spermatozoa were shown to be immotile, the motility disturbance being referred to as epididymal dysfunction. To define the physiological substrates of this descriptive phenomenon, caput spermatozoa from 10 epididymides of five patients who had undergone orchiectomy because of prostatic cancer were investigated. These spermatozoa showed severe motility disturbances, and almost all their flagella stained atypically. Attempts to stimulate such spermatozoa by pentoxifylline achieved a slight improvement in motility. However, addition of seminal plasma from fertile donors resulted in a significant improvement in motility, accompanied by an increase in the number of normally stained flagella. Even better results were achieved by incubation with liposomes made from soybean lipids, mainly phosphatidylcholine. It is concluded that poor motility and atypical staining behaviour of human caput sperm tails can at least partly be related to a lack of phospholipids in the flagellar membrane. Substitution of phospholipids may be a therapeutic approach in procedures using caput spermatozoa, although in addition to the improvement of sperm motility, the enhancement of fertilizing capacity remains to be established.

Influence of phospholipid liposomes (PLL) on UVB-induced erythema formation.

Application of liposomal phospholipids (Natipide II) caused a significant decrease in erythema induced by UVB (280-312 mm) in patients with skin types II and III (n = 31). In accordance with findings in other organs, e.g. liver, the present findings suggest that phospholipids are capable of reducing H2O2 formation and/or increasing glutathione synthesis in human skin, thus reducing erythema formation by preventing oxidative stress.

Enhancement of the penetration of dithranol and increase of effect of dithranol on the skin by liposomes.

An assessment was made in healthy skin of dithranol erythema caused by a 10-min occlusive application of 0.5% dithranol (CAS 1143-38-0) in liposomal gel (Natipide II) with and without the addition of 3% salicylic acid, and of 0.5% dithranol in vaseline and in a removable standard ointment base, both with 3% salicylic acid. Both liposomal preparations led to a significant potentiation of the dithranol erythema. The results indicate a strong promotion of the penetration of dithranol by the liposomal gel.

Increase of lipid fluidity and suppression of proliferation resulting from liposome uptake by human keratinocytes in vitro.

The in vitro effects of liposomes on HaCaT human keratinocytes were studied with regard to their uptake, lipid fluidity and proliferation of the cells. Oligolamellar liposomes, prepared from soya bean phospholipids, had a mean size of 150 mm and consisted predominantly of phosphatidylcholine (83%) and phosphatidylethanolamine (10%) and the fatty acids comprised mainly linoleic acid (66%) or other unsaturated fatty acids. After 6 and 24 h of incubation with 1 and 0.1% w/v of liposomal lipids, phase-contrast microscopy revealed marked cytoplasmic vacuolization of the cells. Keratinocytes treated with the liposomes contained aggregations of multilaminated lipid material without delimiting cell membranes. The cellular lipid fluidity (reciprocal of diphenylhexatriene fluorescence polarization P-value) correlated with liposomal concentration and incubation time. A significant elevation of lipid fluidity (P less than 0.05) was observed with 1 and 0.1% liposomes after 1 h of incubation (81.8 +/- 4.7 and 95.7 +/- 1.2% of control P value) and for 0.01% liposomes after 3 h (96.2 +/- 1.5%). Maximum fluidity occurred after 48 h of exposure to 1% liposomes (42.1 +/- 3.1%). Exposure to liposomal lipids for 24 and 48 h resulted in suppressed cell proliferation with 50% inhibition concentrations (IC50), being 0.06% for incorporation of [3H]-thymidine. 0.08% for [14C]-amino-acid incorporation and greater than 1% for protein content per well after 24 h of exposure. The cells were able to proliferate and lipid fluidity returned to normal within 7 days following discontinuation of incubation with liposomal lipids.

Characterization of Peroxidase Secretion and Subcellular Organization of Phanerochaete chrysosporium INA-12 in the Presence of Various Soybean Phospholipid Fractions.

Stimulation of lignin peroxidase production by exogenous phospholipids depends on the composition of the phospholipid fraction prepared by using the Nattermann process. The fraction composed mainly of negatively charged phospholipids (NAT 89) was the most efficient source for exoprotein secretion by Phanerochaete chrysosporium INA-12. The results of biochemical marker assays and ultrastructural morphology determination by electron microscopy were correlated. Activities of succinate dehydrogenase, a mitochondrial marker, and cytochrome c oxidoreductase, an endoplasmic reticulum (ER) marker, were increased 1.3- and 2.2-fold, respectively, in the presence of NAT 89. Electron microscopy observations suggested that the amount of mitochondria and ER in culture containing phospholipids was increased at the optimum day of lignin peroxidase production. Therefore, phospholipids enhanced energetic metabolism of strain INA-12 and markedly modified fungus physiology. Since ER is involved in enzyme synthesis, we suggest that its increased amount in mycelium cultured with NAT 89 is directly associated with the higher production of lignin peroxidase.

Liposomes from soya phospholipids as percutaneous drug carriers. 1st communication: qualitative in vivo investigations with antibody-loaded liposomes.

The penetration behaviour of liposome (prepared from NAT 106)-incorporated proteins was investigated in vivo by use of monoclonal antibodies (MOAB) as model substances on the skin of young pigs. Within 20 min of topical application, an even distribution of liposome-incorporated antibodies through all skin layers could be shown by means of an immunohistochemical stain.

Significance of empty liposomes alone and as drug carriers in dermatotherapy.

The influence of three different liposome formulations (NAT 106, NAT 50, NAT 89), as empty liposomes and loaded with 0.05% betamethasone, on blood circulation in the corium was investigated by means of laser Doppler flowmetry. All three empty liposomes brought about a decrease in circulation. The maximum effect was reached 90 min after a 30-min occlusive application of the test preparations. Loading with betamethasone resulted in a significant increase in circulation after only 15 min. This increase in interpreted as a steroid effect. No comparable symptoms appeared with the application of base cream DAC (an O/W emulsion) or of betamethasone in base cream under identical experimental conditions.

Liposomes from soya phospholipids as percutaneous drug carriers. 2nd communication: quantitative in vivo investigations with radioactively labelled liposomes.

The percutaneous absorption of liposomes (prepared from NAT 106) was investigated with radioactively labelled substances in comparison with percutaneous absorption without liposomes (controls) in vivo on the skin of young pigs. Radioactivity was monitored as a function of time in skin tissue, plasma or blood and in urine. Elevated tissue levels, increased absorption and renal elimination of the various liposomal preparations in comparison to the controls were measured.

Improvement of the gastric tolerance of non-steroidal anti-inflammatory drugs by polyene phosphatidylcholine (Phospholipon 100).

The effect of co-administration with polyene phosphatidylcholine (Phospholipon 100) on the oral gastrotoxicity of various non-steroidal anti-inflammatory drugs (NSAIDs) was studied in the rat. The highly unsaturated phospholipid reduced gastric mucosal lesions measured 3.5 h after oral administration of aspirin, indomethacin, phenylbutazone, diclofenac, piroxicam and sudoxicam to rats which had received a 3 day bread diet followed by 24 h fasting. The extent of reduction of gastrotoxicity varied amongst the individual NSAIDs. Phospholipon 100 also reduced gastric lesions induced by 3 day oral piroxicam and diclofenac administration. A trend towards reduction of oral diclofenac gastrotoxicity was observed following intravenous Phospholipon 100 administration. Phospholipon 100 H (100% saturated phosphatidylcholine) was less effective than Phospholipon 100 in improving acute gastric tolerance to oral phenylbutazone, diclofenac and piroxicam. Administration of the NSAID-Phospholipon 100 combination produced little change in the anti-inflammatory activities of diclofenac on carrageenan paw oedema and diclofenac and piroxicam on adjuvant arthritis in the rat. Combination with Phospholipon 100 offers a novel means for reducing the gastric side-effects of NSAID therapy.

Induction of high titer mouse--antihuman spermatozoal antibodies by liposome incorporation of spermatozoal membrane antigens.

This is the first report of induction of high titer mouse-antihuman spermatozoal antibodies using a spermatozoal antigen incorporated in liposomes. The peptide antigens that were incorporated into pure phosphatidylcholine liposomes are known to react with the naturally occurring antibodies in sera of sterile patients. Electron microscopy and the complement-dependent glucose release tests indicated that the peptides were entrapped in the aqueous phase between the lipid bilayers in the liposome vehicles. This is in contrast to most other liposomal antigens that are expressed on the outer lipid surface. In view of the nontoxic nature of the liposomes these results could be of some interest in immunological fertility regulation trials with appropriate antigens in an allogeneic system.