Does hydration affect intercellular lipid organization in the stratum corneum?
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Animals↗
Biomedical subjects
R O Potts
Publications and source records attributed to R O Potts.
Barrier function of human keratinocyte cultures grown at the air-liquid interface.
Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism.
Air↗
The influence of stratum corneum morphology on water permeability.
The stratum corneum (SC) provides the barrier to water loss for the skin of mammals. A significant body of evidence now exists suggesting that extracellular SC lipids are primarily responsible for this barrier. We have measured the permeability (P) and lag-time (Tlag) for water vapor transport through the SC and found that P is about 1000 times less than the values obtained for most other lipid membranes. In addition, we have measured the water partitioning into the lipid microdomain of the SC using a differential scanning calorimetry technique. These combined data provide an estimate of the diffusion coefficient (D) and diffusion pathlength (delta). The results show that the intrinsic diffusion of water is comparable to values obtained with other lipid membranes. The value obtained for delta, however, is fiftyfold greater than the sample thickness. These results are interpreted in terms of the unique morphology of the SC, where lipids form an extracellular continuum that is highly tortuous. Thus, the exceedingly low permeability of the SC may be due, in large part, to its unique morphology.
Animals↗
Oleic acid: its effects on stratum corneum in relation to (trans)dermal drug delivery.
Calorimetric studies with porcine stratum corneum (SC) have shown that the lipid phase transitions associated with the intercellular bilayers are markedly affected by treatment with oleic acid. Specifically, the transition temperatures (Tm) and cooperativity are reduced, whereas no effect was observed on the endotherm associated with keratin denaturation, suggesting that oleic acid primarily affects the SC lipids. The decrease in the lipid-associated Tm's was further correlated with the amount of oleic acid taken up by the SC. Parallel experiments with silastic implied that the uptake is dependent on the thermodynamic activity of oleic acid in the vehicle itself. The in vitro transport of Piroxicam across human and hairless mouse skin (HMS) was significantly enhanced by oleic acid, as a function of the extent of oleic acid uptake, with an attendant change in Tm. These results emphasize the role of SC lipids in percutaneous absorption. Transport also depended on the donor concentration of ionized drug suggesting that the enhanced transport mechanism cannot be accounted for solely on the principles of the classical pH-partition hypothesis. Accordingly, a model of skin permeability enhancement involving solid-fluid phase separation within the SC lipids is proposed for oleic acid, consistent with the existing phospholipid literature. In conjunction with the use of oleic acid as an enhancer, very soluble hydrophilic salts were recognized as key factors in attaining maximum delivery. Oleic acid uptake, lipid delta Tm, and enhanced drug flux were all found to correlate, exhibiting a bell-shaped curve as a function of the ethanol vehicle concentration. Therefore, uptake and/or DSC experiments are useful for formulating enhanced topical delivery systems.
Administration, Cutaneous↗
Percutaneous penetration enhancement in vivo measured by attenuated total reflectance infrared spectroscopy.
A novel application of attenuated total reflectance IR spectroscopy (ATR-IR) was used to monitor the outer several microns of the stratum corneum (SC) and, thereby, demonstrate enhanced percutaneous absorption in vivo in man. 4-Cyanophenol (CP) as a model permeant yielded a unique IR signal, distinct from those of the stratum corneum and the vehicle components. CP was administered for 1, 2, or 3 hr as a 10% (w/v) solution either in propylene glycol or in propylene glycol containing 5% (v/v) oleic acid. The absorbance at 2230 cm-1, which corresponded to C identical to N bond stretching, diminished significantly faster when CP was codelivered with oleic acid. An IR absorbance due primarily to propylene glycol at 1040 cm-1 (C-O stretching) also disappeared more quickly following application of the enhancer-containing solution. In addition, only the formulations with oleic acid induced a higher wavenumber shift in the frequency of the asymmetric C-H bond stretching absorbance. This change indicates increased lipid-chain disorder, the mechanism by which oleic acid is believed to cause enhanced drug transport across the stratum corneum. Therefore, ATR-IR permits one to examine noninvasively the kinetics, extent, and mechanism of percutaneous penetration enhancement in vivo in human subjects.
Adult↗
Lipid biophysics of water loss through the skin.
The regulation of water loss through the skin is a poorly understood but crucial process in maintaining terrestrial life-forms. In mammalian skin, the outermost layer, called the stratum corneum (SC), is rate-limiting to water loss. We have evaluated temperature-dependent changes in water vapor permeability and infrared spectra of porcine SC. In particular, we have analyzed the infrared absorption peaks due to the extracellular lipids of the SC. These results show a remarkable correlation between water permeability and the frequency of the C-H stretching vibrations over a broad range of temperature. Since the spectral changes reflect an increased number of alkyl gauche conformers, these results suggest that water permeability is dependent upon the hydrocarbon-chain disorder of SC lipids.
Animals↗
Examination of stratum corneum barrier function in vivo by infrared spectroscopy.
It is generally accepted that the stratum corneum (SC) is the least permeable layer of the epidermis. Histologically, though, the SC is a non-uniform, inhomogeneous membrane, and the question "Is barrier function distributed uniformly across the SC thickness?" has been posed. To address this issue, human ventral forearm SC has been studied in vivo by attenuated-total-reflectance Fourier-transform infrared spectroscopy during the course of sequential tape-stripping. Because the intercellular lipids of the SC and the degree of hydration of the membrane have been shown to be crucial determinants of barrier function, attention has been focused on the spectral features, which report specifically on these parameters. The degree of disorder of the SC intercellular lipids has been found to decrease over the outer cell layers (up to three tape-strips) and then to remain essentially constant. The amount of lipids decreases similarly such that a 60% reduction (relative to the "no-strip" baseline) is observed after about four tape-strips. A plausible explanation for these measurements is that the lipids near the surface are a mixture of (a) "true" intercellular lipid (which is expected to be highly ordered), and (b) sebaceous lipid (which contains much greater amounts of low-melting components, such as fatty acids). The sequential infrared (IR) spectra provide at least circumstantial evidence to support this hypothesis. As expected, the IR spectra show that SC hydration increases from the surface towards the SC-stratum granulosum interface. Taken together, the results imply that the SC is indeed non-uniform. The properties of the outer layers (those removed by the first 3-4 tape-strips) change significantly with increasing depth.(ABSTRACT TRUNCATED AT 250 WORDS)
Epidermal Cells↗
Transdermal drug transport and metabolism. I. Comparison of in vitro and in vivo results.
Using excised human skin and tissue grafted to athymic mice, the in vitro and in vivo delivery and metabolism of a salicylate diester were compared. Concentration profiles of this drug and its metabolites were obtained for the outer several hundred microns of the skin. These results show significant differences in the extent of enzymatic cleavage and distribution of metabolites between in vitro and in vivo studies. Furthermore, these data suggest that in vitro results may overestimate metabolism because of increased enzymatic activity and/or decreased capillary removal.
Administration, Cutaneous↗
Transdermal drug transport and metabolism. II. The role of competing kinetic events.
The steady-state flux and skin tissue distribution of a topically applied diester of salicylic acid was measured in vitro in the presence and absence of an esterase inhibitor. When compared with data obtained previously under in vivo conditions, the results presented here provide insight into the role of competing diffusional and hydrolytic events in the delivery and distribution of topically applied drugs. Furthermore, these results, when combined with a theoretical analysis of topical drug delivery and metabolism, suggest that the differences in delivery and drug distribution seen under in vitro conditions are related to both altered drug removal and altered hydrolysis rates.
Administration, Cutaneous↗
The water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface.
In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.
Animals↗
Stratum corneum lipid phase transitions and water barrier properties.
In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.
Animals↗
Role of stratum corneum lipid fluidity in transdermal drug flux.
Fatty acids are effective penetration enhancers for the transdermal delivery of certain co-applied drugs. In order to assess the mechanism of enhancement, spectrometric, calorimetric, and flux techniques were used to study porcine stratum corneum following treatment with a series of cis- and trans-positional isomers of octadecenoic acid. Results obtained from spectrometric and calorimetric measurements show increased lipid fluidity following treatment of the stratum corneum with the cis monoenoic acids which have the site of unsaturation centrally located. Under similar conditions, these same cis monounsaturated acids resulted in enhancement of salicyclic acid flux through porcine skin. The striking parallelism between flux and fluidity measurements suggests that transdermal drug flux may be ultimately related to stratum corneum lipid structure.
Animals↗
Lipid thermotropic transitions in human stratum corneum.
The techniques of differential scanning calorimetry (DSC) and thermal perturbation infrared (IR) spectrometry were used to investigate thermal transitions in intact, fractionated, and lipid-extracted human stratum corneum. The DSC results show 3 major and one minor thermal transition in the range of 30-120 degrees C. Of particular interest to this study are 2 transitions seen near 65 degrees and 75 degrees C in intact stratum corneum and a stratum corneum membrane preparation, but absent from lipid-extracted samples. Results of IR spectrometry show that thermally induced spectral changes related to enhanced motion of the lipid acyl chains also occur in the region of about 60-80 degrees C. The combined DSC and IR results show that the thermal transitions near 65 degrees and 75 degrees C depend on water concentration in a manner identical to that seen for a variety of lipid-water systems. Taken together, these results suggest that thermal transitions occurring near 65 degrees and 85 degrees C involve increased acyl chain motion of stratum corneum lipids.
Calorimetry, Differential Scanning↗
A noninvasive, in vivo technique to quantitatively measure water concentration of the stratum corneum using attenuated total-reflectance infrared spectroscopy.
In order to noninvasively measure water concentration in the stratum corneum, infrared spectra were obtained using an attenuated total-reflectance technique in conjunction with Fourier transform spectroscopy. A weak water-absorbance band near 2,100 cm-1 was detected in both in vivo and in vitro spectra. The significance of this band is that it occurs in a region of the mid-infrared where the stratum corneum and most topically applied substances show no absorbance. In vitro spectra obtained as a function of ambient relative humidity showed an increase in the absorbance near 2,100 cm-1 with increasing water concentration in the stratum corneum. The combined in vivo and in vitro results lead to a quantitative assessment of water concentration in the uppermost layers of the stratum corneum.
Animals↗
Changes with age in the moisture content of human skin.
A technique to measure the dynamic mechanical properties of human skin in vivo is described. The technique measures the propagation and attenuation of shear waves in skin tissue over a range of frequencies (8-1016 Hz). Results show that both the propagation velocity and attenuation of shear waves in skin are highly dependent upon the water content of the stratum corneum. The technique was used to measure the dynamic mechanical properties of the skin on the back of the left hand for a group of 16 men ranging in age from 24-63 years. The results suggest that aged skin has a lower water content than the skin of younger men.
Adult↗
The dynamic mechanical properties of human skin in vivo.
The propagation and attenuation of shear waves in human skin is investigated over a frequency range of near zero to 1000 Hz. The results show that at frequencies below a few hundred Hz, shear waves propagate along the skin surface and thus provide information on the dynamic mechanical properties of the stratum corneum. The surface waves, however, are rapidly attenuated with increasing frequency so that at frequencies above about 500 Hz the energy is propagated only as bulk shear waves. Thus, at high frequencies the properties measured are those of the dermis.
Biomechanical Phenomena↗
Effects of magnesium and ionic strength on the diffusion and charge properties of several single tRNA species.
The technique of laser light scattering was used to evaluate the effects of Mg+2 and ionic strength on the solution structures of seven tRNA species. Information about ion effects on both conformation and electric charge were derived from measurements of the translational diffusion constants and diffusive virial coefficients. E. coli tRNAMetf and six elongator tRNAs from both Class I and II were studied. The diffusion measurements show that the responses of all but the initiator species are qualitatively similar to each other and to that of bulk tRNA, but that significant quantitative differences also obtain. All of the elongator species exhibited an anomolous increase in diffusivity reported earlier by us for bulk tRNA when placed in a low salt-low Mg+2 condition. The initiator tRNA did not undergo this transition and unlike the other tRNAs tested was apparently more compact in 1 mM Mg+2 than 10 mM Mg+2 at ionic strengths in excess of 0.1 M. At 0.1 M ionic strength, pH 7.2, the average net charge of the tRNAs ranged from 7-12 e- in 1 mM Mg+2 and 3-7 e- in 10 mM Mg+2, consistent with the binding of 1-2 additional Mg+2 ions in the higher Mg+2 condition.
Escherichia coli↗
Changes in the solution structure of yeast phenylalanine transfer ribonucleic acid associated with aminoacylation and magnesium binding.
The effect of aminoacylation on the structure of yeast phenylalanine tRNA was evaluated by laser light scattering. In these experiments, the translational diffusion coefficient (D20,w) of phenylalanyl-tRNA was monitored continuously during spontaneous deacylation in a variety of solution conditions. The results reveal that significant changes can occur in the hydrodynamic volume and electric charge as a consequence of aminoacylation but that the effects are magnesium dependent. At neutral pH, 20 degrees C, and 0.1 M salt, the D20,w value increased by 18% when deacylation occurred in 2--10 mM Mg2+ concentrations while no change in diffusivity was observed for tRNA deacylating in 0.5--1.0 mM Mg2+. The Mg2+ concentration dependence of the D20,w changes behaves in highly cooperative manner. The electric charges of aminoacyl-tRNA and nonacylated tRNA in 1 and 10 mM Mg2+ were estimated from the diffusive virial coefficients. In the higher Mg2+ conditions, aminoacyl-tRNA has a charge of 15 +/- 2e- while that of the nonacylated form is 10 +/- 2e-; both acylated and nonacylated tRNA have a charge of 11 +/- 4e- in 1 mM Mg2+. Taken together, the results indicate that aminoacylation permits the binding of additional Mg2+, resulting, in turn, in the formation of a more extended conformer of lower diffusivity and greater negative charge. The results also provide a possible explanation for several contradictory results in the literature.
Amino Acyl-tRNA Synthetases↗