A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.     

Influence of fatty acids on the binding of calcium to human serum albumin

Fatty acids have been shown to influence the binding of calcium to human serum albumin. The calcium binding to albumin was enhanced when long-chain fatty acids were added to albumin prepared by two different methods and decreased when fatty acids were removed from albumin. Palmitic, stearic and oleic acids all exhibited this phenomenon. The effects of long-chain fatty acids on the binding of calcium to albumin in vitro appears to be of sufficient magnitude to have in vivo implications in calcium homeostasis and in determining the ratio of free to total calcium. Preliminary in vivo experiments have confirmed the calcium binding of fatty acids in serum and suggest a physiological role for this phenomena.     

A new colorimetric method for the determination of free fatty acids with acyl-CoA synthetase and acyl-CoA oxidase

A rapid and sensitive spectrophotometric assay for free fatty acids using acyl-CoA synthetase and acyl-CoA oxidase is described. It is sensitive to as low as 5 nmol of free fatty acids, and the standard curve is linear up to 100 nmol. The assay consists of the measurement of H2O2 produced from free fatty acids by acyl-CoA synthetase and acyl-CoA oxidase. The quantity of H2O2 is determined by the absorbance at 550 nm in the presence of catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (AHMT). This method shows a broad specificity to long-chain fatty acids and the recoveries of added fatty acids (C12-C18) are more than 90%. The presence or absence of serum components or Escherichia coli cell-free extracts has no significant effect on the recovery of added palmitic acid.     

Transport of activated fatty acids by the peroxisomal ATP-binding-cassette transporter Pxa2 in a semi-intact yeast cell system

In the yeast Saccharomyces cerevisiae, fatty acid beta-oxidation is restricted to peroxisomes. Previous studies have shown two possible routes by which fatty acids enter the peroxisome. The first route involves transport of medium-chain fatty acids across the peroxisomal membrane as free fatty acids, followed by activation within the peroxisome by Faa2p, an acyl-CoA synthetase. The second route involves transport of long-chain fatty acids. Long-chain fatty acids enter the peroxisome via a route that involves activation in the extraperoxisomal space, followed by transport across the peroxisomal membrane. It has been suggested that this transport is dependent upon the peroxisomal ATP-binding-cassette transporters Pxa1p and Pxa2p. In this paper we investigated whether Pxa2p is directly responsible for the transport of C18:1-CoA, a long-chain acyl-CoA ester. Using protoplasts in which the plasma membrane has been selectively permeabilised by digitonin, we show that C18:1-CoA, but not C8:0-CoA, enters the peroxisome via Pxa2p, in an ATP-dependent fashion. The results obtained may contribute to the elucidation of the primary defect in the human disease X-linked adrenoleukodystrophy.     

A new peroxisomal beta-oxidation disorder in twin neonates: defective oxidation of both cerotic and pristanic acids

Twin brothers were born with clinical symptoms indicating that they were suffering from Zellweger syndrome. However, instead of a generalized peroxisomal dysfunction, only very long-chain fatty acids and the pristanic acid/phytanic acid ratio were elevated in plasma and decreased oxidation of very long-chain fatty acids and pristanic acid was the only impairment found in fibroblasts. The other peroxisomal parameters tested were normal, including normal oxidation of phytanic acid and normal activity of dihydroxyacetonephosphate acyltransferase in fibroblasts as well as normal plasma bile acids. Although the biochemical results point to a defect in peroxisomal beta-oxidation, the isolated finding of impaired oxidation of very long-chain fatty acids and pristanic acid has to our knowledge not been reported previously and is difficult to explain by a deficiency of a known peroxisomal beta-oxidation enzyme.     

Direct evidence of methylglucose lipopolysaccharides/palmitoyl-CoA noncovalent complexes by capillary zone electrophoresis-electrospray/mass spectrometry

Mycobacterial methylglucose lipopolysaccharides (MGLPs) play an important regulatory role in the biosynthesis of long-chain fatty acids by forming complexes with neosynthesized acyl-CoA fatty acid derivatives. The MGLPs from Mycobacterium smegmatis were purified by high-performance anion-exchange chromatography and characterized by LSIMS and CE/ESI-MS. We investigated their interaction with palmitoyl-CoA using capillary zone electrophoresis with both direct and indirect UV detection. In the latter mode, the signal of the UV-transparent MGLPs decreased upon addition of increasing amounts of palmitoyl-CoA; while using direct UV detection, the addition of palmitoyl-CoA to the MGLPs revealed characteristic profiles. The major peak was assigned to the noncovalent MGLP/palmitoyl-CoA complex on the basis of its electrophoretic mobility. The abundance of the complex was found to increase until the MGLP/palmitoyl-CoA molar ratio reached a 1/1 stoichiometry. The existence of and the stoichiometry of this complex were assessed by CE/ESI mass spectrum analysis, showing pseudomolecular ions of the MGLP/palmitoyl-CoA complex. These results confirm that CE/ESI-MS is a powerful tool to characterize noncovalent molecular association.     

Lipid modification strategies in the production of nutritionally functional fats and oils

Rapid improvements in the understanding of the nutritional requirements of both infants and adults has led to new developments in the modification of fats and oils. Specific targets include the improvement in growth and development of infants, treatment of disease in adults, and disease prevention. Efforts have been focussed on the production of structured lipids using medium-chain acids and long-chain polyunsaturated fatty acids (PUFAs), as well as the concentration of long-chain PUFAs from new and existing sources. Short- and medium-chain fatty acids are metabolized differently than long-chain fatty acids and have been used as a source of rapid energy for preterm infants and patients with fat malabsorption-related diseases. Long-chain PUFAs, specifically docosahexaenoic acid and arachidonic acid, are important both in the growth and development of infants, while n-3 PUFAs have been associated with reduced risk of cardiovascular disease in adults. Based on the requirements for individual fat components by different segments of the population, including infants, adults, and patients, ideal fats can be formulated to meet their needs. By using specific novel fat sources and lipid modification techniques, the concentrations of medium-chain, long-chain saturated, and long-chain polyunsaturated fatty acids as well as cholesterol can be varied to meet the individual needs of each of these groups. While genetic modification of oilseeds and other novel sources of specific lipid components are still being developed, chemical and lipase-catalyzed interesterification reactions have moved to the forefront of lipid modification technology. Fractionation of fats and oils to provide fractions with different nutritional properties has potential, but little work has been performed on the nutritional applications of this method. The choice of suitable lipid modification technologies will depend on the target lipid structure, production costs, and consumer demand. A combination of some or all of the present lipid modification techniques may be required for this purpose.     

Fatty acid composition of myelin proteolipid protein during vertebrate evolution

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.     

Corandomization of fats improves absorption in rats

Human milk fat is well absorbed despite its large concentration of long-chain saturated fatty acids (LCSFA), particularly palmitic acid. The superior absorption has been ascribed in part to the uncommonly high proportion of the palmitic acid in the sn-2 position of the triglycerides, 70% in human milk triglycerides compared with 10-15% in the fats and oils commonly used in infant formula blends. Colipase-dependent pancreatic lipase selectively hydrolyzes the fatty acids at the sn-1 and 3 positions, yielding free fatty acids and the 2-monoglyceride, which are absorbable. Free palmitic acid, but not monopalmitin, can be lost as calcium soaps in the feces. The present study demonstrated that mixtures of coconut oil and palm olein are better absorbed by rats if the proportion of LCSFA in the sn-2 position is increased by the process of chemical randomization, in which the fatty acids of the native oils are redistributed equally to all three positions in the rearranged triglycerides. The fecal excretion of total fatty acids, essentially LCSFA, from the mixtures in which the oils were randomized together (corandomized) was 30 to 60% less than from the mixtures of native oils.     

Sustaining the development of primary care in academic medicine. Working Group on Sustaining the Development of Academic Primary Care. Association of American Medical Colleges

Dietary supplementation of very long-chain n-3 fatty acids to rats reduces postprandial plasma concentrations of triacylglycerol, unesterified fatty acids and glycerol after long-term feeding by unknown mechanisms [Rustan et al., J. Lipid Res. 34 (1993) 1299-1309]. In the present study we examine the role of adipose tissues in metabolism of fatty acids. Postprandial plasma concentrations of triacylglycerol, unesterified fatty acids and glycerol were reduced by 75%, 50% and 30%, respectively, during 49 days of feeding high-fat diets containing n-3 fatty acids (6.5% n-3 fatty acid concentrate, 13% lard) as compared to lard (19.5% lard). These differences were observed already after two days of feeding. Plasma concentration of unesterified very long-chain n-3 fatty acids increased to 50 microM in n-3 fatty acid-supplemented rats, whereas these fatty acids were undetectable in lard-fed animals. The n-3 fatty acid-enriched diet limited cell volumes of perirenal and epididymal adipocytes by 40% and 30%, respectively, after 49 days, as compared to lard feeding. This reduction in cell volume was not due to reduced synthesis of glycerolipids in epididymal adipocytes. Acute incubation of perirenal and epididymal adipocytes with oleic acid or eicosapentaenoic acid, caused similar increase in synthesis of triacylglycerol. Dietary supplementation with n-3 fatty acids decreased basal and total lipolysis (isoprenalin-stimulated) in perirenal adipocytes. Basal lipolysis in epididymal adipocytes was reduced by n-3 fatty acids only after 49 days. n-3 fatty acids increased total lipolysis in mesenteric and subcutaneous fat cells compared to adipocytes derived from lard-fed animals, whereas basal lipolysis was unchanged. These results suggest that the reduced postprandial plasma concentration of unesterified fatty acids after n-3 fatty acid-supplementation is not caused by accumulation of fatty acids in adipose tissue. The reduced trophic growth of adipocytes might be due to decreased supply of unesterified fatty acids for triacylglycerol storage. (c) 1998 Elsevier Science B.V.     

High rates of [1-14C]acetate incorporation into the lipid of isolated spinach chloroplasts

Spinach chloroplasts, isolated by techniques yielding preparations with high O2- evolving activity, showed rates of light-dependent acetate incorporation into lipids 3-4 fold higher than any previously reported. Incorporation rates as high as 500 nmol of acetate/h per mg of chlorophyll were measured in buffered sorbitol solutions containing only NaHCO3 and [1-14C]acetate, and as high as 800 nmol/h per mg of chlorophyll when 0.13 mM-Triton X-100 was also included in the reaction media. The fatty acids synthesized were predominantly oleic (70-80% of the total fatty acid radioactivity) and palmitic (20-25%) with only minor amounts (1-5%) of linoleic acid. Linolenic acid synthesis was not detected in the system in vitro. Free fatty acids accounted for 70-90% of the radioactivity incorporated and the remainder was shared fairly evenly between 1,2-diacylglycerols and polar lipids. Oleic acid constituted 80-90% of the free fatty acids synthesized, but the diacylglycerols and polar lipids contained slightly more palmitic acid than oleic acid. Triton X-100 stimulated the synthesis of diacylglycerols 3-6 fold, but stimulated free fatty acid synthesis only 1-1.5-fold. Added glycerol 1-phosphate stimulated both the synthesis of diacylglycerols and palmitic acid relative to oleic acid, but did not increase acetate incorporation into total chloroplast lipids. CoA and ATP, when added separately, stimulated acetate incorporation into chloroplast lipids to variable extents and had no effect on the types of lipid synthesized, but when added together resulted in 34% of the incorporated acetate appearing in long-chain acyl-CoA. Pyruvate was a much less effective precursor of chloroplast fatty acids than was acetate.     

Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin

Fluorescent fatty acid labels have been incorporated into the palmitoylation sites of rhodopsin and used to probe the membrane accessibility and location of these sites. The fluorescence properties of anthroyloxy and pyrenyl fatty acids bound to rhodopsin were investigated in a reconstituted vesicle system. Collisional quenching of fluorescence by stearic acid (DSA) labeled with doxyls in the 16, 12, and 5 positions was used to determine the membrane accessibility and disposition of the modifying fatty acids. To properly determine the membrane concentration of these quenchers, the dependence of the Stern-Volmer parameters on both quencher and vesicle concentration was determined. An analysis of these dependences provided a correction for partitioning of the quencher between the aqueous phase and the membrane. After this correction, the relative effectiveness of doxyl quenchers was 16-DSA > 12-DSA > 5-DSA. Parallel studies on free anthroyloxy and pyrenyl fatty acids incorporated into the reconstituted system showed the same dependence on quencher position. These results indicate that the labels at the palmitoylation sites of rhodopsin are situated in the membrane much as a free fatty acid. This anchoring of the palmitates in the membrane results in the formation of a fourth cytoplasmic loop.     

Ca2+ induces an increase in cGMP-phosphodiesterase activity in squid retinal photoreceptors

In this paper we describe a rapid, isocratic high performance liquid chromatography (HPLC) method for the study of radioactive fatty acid incorporation into complex lipids of human erythrocytes, which allows the simultaneous separation of the major phospholipid classes and long-chain acylcarnitines. The lipid extract of erythrocytes pulsed with radioactive fatty acids was injected into an HPLC system equipped with a silica column. The individual components eluted were monitored by ultraviolet absorption and radioactive emission. With respect to the UV profile, the radioactive profile showed an additional peak between phosphatidyl-choline and phosphatidylethanolamine, which was identified as long-chain acylcarnitine by different experimental approaches. The radioactivity recovered in the long-chain acylcarnitines contains essential information enabling definition of acyl trafficking in red cells.     

Elaboration of the messenger transport organizer pathway for localization of RNA to the vegetal cortex of Xenopus oocytes

1. Two experiments were designed to study the influence of free fatty acid content and degree of saturation of free fatty acids and neutral fat on digestibility of added fats and fatty acids. Sunflower oil and tallow were used as neutral fats, and palmitic, stearic, oleic and linoleic acids as free fatty acids. Fat inclusion was 80 g/kg and mixtures of each fat and each free fatty acid were prepared in the proportions 100:0, 70:30 and 40:60. 2. Experimental diets were evaluated for fat and fatty acid digestibilities with broiler chickens at 21 d of age. The metabolisable energy of fat was calculated from the product of digestibility and gross energy. Increasing concentrations of saturated free fatty acids decreased the ME of added fat, whereas unsaturated free fatty acids did not significantly affect the ME value of added fat. 3. Digestibilities of individual fatty acids were analysed by linear regression with rate of inclusion of free fatty acid in the fat blend: palmitic and stearic acids gave a negative slope, whereas oleic and linoleic acids gave a slope not statistically different from zero. Because slopes for saturated fatty acids did not differ between the sunflower oil and tallow treatments, synergism between unsaturated and saturated fatty acids was not detected.     

Microfluorometry using fluorescein diacetate reflects the integrity of the plasma membrane in UVA-irradiated cultured skin fibroblasts

Hepatic coenzyme A (CoA) plays an important role in cellular lipid metabolism. Because mitochondria and peroxisomes represent the two major subcellular sites of lipid metabolism, the present study was designed to investigate the specific impact of hepatic CoA deficiency on peroxisomal as well as mitochondrial beta-oxidation of fatty acids. CoA deficiency (47% decrease in free CoA and 23% decrease in total CoA) was produced by maintaining weanling male Sprague-Dawley rats on a semipurified diet deficient in pantothenic acid (the precursor of CoA) for 5 weeks. Hepatic mitochondrial fatty acid oxidation of short-chain and long-chain fatty acids were not significantly different between control and CoA-deficient rats. Conversely, peroxisomal beta-oxidation was significantly diminished (38% inhibition) in livers of CoA-deficient rats compared to control animals. Peroxisomal beta-oxidation was restored to normal levels when hepatic CoA was replenished. It is postulated that since the role of hepatic mitochondrial beta-oxidation is energy production while peroxisomal beta-oxidation acts mainly as a detoxification system, the mitochondrial pathway of beta-oxidation is spared at the expense of the peroxisomal pathway when liver CoA plummets. The present study may offer an animal model to investigate mechanisms involved in peroxisomal diseases.     

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity

We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.     

Biocatalyst engineering by assembly of fatty acid transport and oxidation activities for In vivo application of cytochrome P-450BM-3 monooxygenase

The application of whole cells containing cytochrome P-450BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to omega-1, omega-2, and omega-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the beta-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.     

Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta

The aim of this study was to investigate location and the types of membrane-associated and cytoplasmic fatty acid-binding proteins in human placental trophoblasts using monospecific polyclonal antibodies. Western blot analysis demonstrated the presence of multiple membrane and cytoplasmic fatty acid transport/binding proteins in human placenta. In addition to previously reported placental membrane fatty acid-binding (p-FABPpm, 40 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transport protein (FATP, 62 kDa) were detected in both microvillous and basal membranes of the human placenta. Among the cytoplasmic proteins, heart (H) and liver (L) type FABP were detected in the cytosol of the human placental primary trophoblasts as well as in human placental choriocarcinoma (BeWo) cells. The immunoreactivity of epidermal type (E)-FABP was not detected in trophoblasts or BeWo cells despite its presence in human placental cytosol. Location of FAT and FATP on the both sides of the bipolar placental cells may favour transport of free fatty acids (FFA) pool in both directions i.e. from the mother to the fetus and vice versa. However, p-FABPpm, because of its exclusive location on the microvillous membranes, may favour the unidirectional flow of maternal plasma long-chain polyunsaturated fatty acids present in the FFA pool to the fetus, due to binding specificity for these fatty acids. Although the roles of these proteins in placental fatty acid uptake and metabolism are yet to be understood fully, their complex interaction may be involved in the uptake of maternal FFA by the placenta for delivery to the fetus.     

Digestion and absorption of dietary triglycerides

This review is an attempt to put in order some facts obtained during twenty-five years of studying the digestion and absorption of dietary triglycerides. After a short history of this problem to show the progress of research, the active mechanism of pancreatic lipase on triglycerides is explained: this enzyme specifically hydrolyses fatty acids esterified with the primary hydroxyl groups of glycerol, forming 2-monoglycerides and free fatty acids. This fact is very important for the further process involving formation and composition of intraluminal micelles, along with absorption and de novo synthesis of triglycerides inside enterocytes. Several different points are disscussed: - Short or medium chain-length fatty acids are hydrolyzed more easily than long-chain ones; after their absorption, the former go to the liver by the portal circulation and the latter go to lymph. - An evaluation of the extent of digestive hydrolysis has been given by means of doubly-labelled triglycerides (glycerol and fatty acids), and by comparing isotope ratio values of lymph and dietary triglycerides. - The physico-chemical state that intraluminal fats are in is discussed; 2-monoglycerides and fatty acids released from triglyceride hydrolysis form macromolecular agregates with biliary salts and phospholipids, cholesterol, and dietary phospholipids (or their degradation products). These are termed mixed micelles and are absorbed by diffusion. - Many recent morphological studies carried out with the electron microscope are indicated. - Two metabolic pathways for triglyceride resynthesis in the mucosal cells have been established, one from free fatty acids and alpha-glycerophosphate, the other from 1 or 2-monoglycerides. Phosphatidic acids are involved in this resynthesis, but the role of other phospholipids is almost certainly not so narrowly limited. - The apparent digestibility of fats according to their fatty acid composition, and mainly according to the location of the fatty acids in the initial triglyceride molecule, is discussed. - The nature and importance of endogenous fatty acids in the digestive tract and lymph is discussed. - The lipid and fatty acid composition of lymph chylomicrons is given. - The importance of lymph lipid particles (average diameter 0,05 less than or equal mum) for transport of exogenous fatty acids is indicated. The biochemical mechanism involved in the digestion and absorption of dietary lipids are now well known, but some processes are still uncertain, particularly the physico-chemical state of intraluminal lipids, the role of enterocyte membranes, the synthesis and secretion of digestive enzymes, and the role of intestinal flora.     

Paired pulse analysis of ATP and noradrenaline release from sympathetic nerves of rat tail artery and mouse vas deferens: effects of K+ channel blockers

The barrier function of the skin resides in the stratum corneum (SC). This outermost layer consists of protein-rich corneocytes and lipid-rich intercellular domains. These domains form the rate-limiting step for transepidermal water loss and the penetration of substances from the environment. To study the nature of the barrier function, stratum corneum lipid models have been examined with wide-angle X-ray diffraction. A disadvantage of this technique is that it requires bulk quantities of lipid and thus information on variations in the lateral packing cannot be obtained in the microm-range. To the best of our knowledge, this is the first study in which electron diffraction is applied on SC lipid model systems. Using this technique, local structural information was obtained about mixtures prepared from isolated pig ceramides, cholesterol, and long-chain free fatty acids. It appeared that addition of free fatty acids caused a transition from a hexagonal to an orthorhombic packing and that electron diffraction can be applied to distinguish between these two lattices. The results are in good agreement with wide-angle X-ray diffraction data and suggest that application of electron diffraction in skin studies can provide new information on the lipid organization in well-defined areas of the stratum corneum.