Mammalian stratum corneum contains physiologic lipid thermal transitions.

Using a new high-sensitivity differential scanning calorimeter, capable of very slow scanning rates and large sample volumes, we examined the thermal transitions in neonatal mouse stratum corneum. Both physiological and supraphysiological transitions were found in intact tissue that were displaced on cooling and obliterated by solvent treatment establishing them as lipids. Physiologic peaks were encountered in lipid extracts from the same tissues. With heating and cooling recycling we found a novel effect of thermal "fractionation" of the peaks into discrete subfractions that appeared to correspond roughly the the number of bands found on thin-layer chromatography of the lipid extracts.

Calorimetric and electron spin resonance examination of lipid phase transitions in human stratum corneum: molecular basis for normal cohesion and abnormal desquamation in recessive X-linked ichthyosis.

Lipids appear to play a critical role as regulators of stratum corneum desquamation. In this study, we observed discrete lipid phase transitions at physiologic temperatures in both normal human scale (NHS) and in lipid extracts of NHS by differential scanning calorimetry. In contrast, such thermal transitions were not observed in recessive x-linked ichthyosis scale (RXLIS). To gain further insight into the molecular basis of the lipid phase transitions in NHS vs. RXLIS, comparable samples were evaluated by electron spin resonance, utilizing the perdeuterated probe, di-t-butyl nitroxide. Upon electron spin resonance analysis, both NHS and RXLIS demonstrated thermal phase transitions in the physiologic range; however, the nature of the lipid environments in each type varied. Whereas the environment of the spin probe was more polar in NHS than in RXLIS, the spin probe partitioned into a more "fluid" environment in RXLIS; i.e., the spin probe was more mobile in RXLIS than in NHS lipid matrices. Because an alteration in the cholesteryl sulfate:cholesterol ratio is the primary lipid abnormality in RXLIS, model cholesterol-fatty acid-cholesteryl sulfate mixtures were prepared in proportion to the lipid composition of NHS and RXLIS. Differences were observed in both thermal transitions and in lipid microenvironments in these mixtures that paralleled those observed in scale samples. Based on these results, a model is proposed that invokes abnormal hydrogen bonding, due to increased cholesteryl sulfate, as the mechanism for the abnormal desquamation in recessive X-linked ichthyosis.

Localization of lipid microdomains and thermal phenomena in murine stratum corneum and isolated membrane complexes: an electron spin resonance study.

The intercellular region of the stratum corneum can be isolated in the form of membrane complexes (intercellular lipids and adjacent cornified envelopes) which are devoid of the cytoplasmic components. In this study the temperature induced phase transitions and corresponding lipid domain reorganization in isolated stratum corneum (SC) sheets and SC membrane complexes (SCM) were determined using the electron spin resonance (ESR) spin probe technique. The spin probe perdeuterated di-tert-butyl nitroxide (pdDTBN) in SC and SCM revealed less well-defined physiologic phase transitions in the SCM and a more polar lipid domain in SC. However, the ESR results show the coexistence of highly ordered domains (immobilized spin probe) and less viscous domains in intact SC, which persist in SCM. Above approximately 20 degrees C the spin probe is dissolved in a highly disordered (isotropic) lipid domain in both SC and SCM. In both solvent extracted SC and SCM, the spin probe is dissolved in a highly ordered lipophilic domain associated with the lipids complexed to the corneocyte envelope and exhibiting no thermal transitions between -23 degrees to 60 degrees C. Further, the amount of mobile spin probe is related to the amount of residual lipid. An unexpected finding was the apparent reduction of the spin probe in solvent extracted SCM, suggesting the presence of a previously unrecognized free radical reducing mechanism in these sites. The mobilities of the spin probe when dissolved in model lipids, non-hydroxy, and hydroxy containing ceramides and cholesteryl oleate, differed significantly from those observed in SC or SCM. These studies demonstrate the usefulness of ESR for the localization and characterization of lipid microenvironments in the stratum corneum.

Effect of organic solvents on in vitro human skin water barrier function.

Skin barrier disruption caused by organic solvents to human cadaver dermatomed skin was evaluated using an in vitro model system. Resultant changes in transepidermal water loss (TEWL), as measured with an evaporimeter, were recorded after topical application of either acetone, chloroform:methanol 2:1, hexane, hexane:methanol 2:3, or the control, water, for exposure times of 1, 3, 6, and 12 min. The resultant lipid/solvent mixture was removed and analyzed for its lipid content. The ability of the different solvents to induce changes in the skin's barrier function was assessed by comparing pre- to post-solvent exposure TEWL (delta TEWL). When compared to the controls, water and unexposed skin, chloroform:methanol 2:1 caused the greatest significant increase in TEWL, followed by hexane:methanol 2:3. Acetone and hexane showed no difference in TEWL from the controls. Besides solvent, exposure time was a significant independent variable for predicting delta TEWL, and the interaction of the two (exposure time and solvent type together) was the strongest predictor. Lipid analysis of the extracts revealed that all the solvents removed comparable quantities of the surface lipids (triglycerides, wax esters, squalene, cholesterol esters). Stratum lipids--ceramides, free fatty acids, and cholesterol--extracted by chloroform:methanol 2:1 and hexane:methanol 2:3 were comparable and significantly greater than those extracted by acetone and hexane. These two solvents failed, however, to induce comparable changes in TEWL, as chloroform:methanol 2:1 induced a significantly greater delta TEWL than hexane:methanol 2:3. Additionally, no individual lipid class extracted by either chloroform:methanol 2:1 or hexane:methanol 2:3 proved to be a significant or accurate variable for predicting delta TEWL. This suggests that the mechanism by which topical chloroform:methanol 2:1 and hexane:methanol 2:3 exposure induce a delta TEWL involves more than pure lipid extraction.

Iodine deficiency produces hypercalcemia and hypercalcitonemia in rats.

To determine the effect of thyroid-stimulating hormone (TSH) on secretion of calcitonin by the thyroid, 50 male Sprague-Dawley rats were randomly separated into seven groups. The groups received different diets, medications, or operations [propylthiouracil (PTU), iodine-deficient diet, (LID), acute or chronic thyroxine treatment, sham operation (SO), hemithyroidectomy (Htx), and total thyroidectomy (Ttx)]. two weeks to six months later, serum TSH concentrations were increased in the Htx, Ttx, and LID groups when compared with SO animals. Serum calcitonin concentrations were increased in the LID- and PTU-treated groups and were decreased in animals that chronically received thyroxine. Serum calcium concentrations were increased in the LID animals, decreased in the Ttx animals, and were similar in the other groups. These findings suggest that TSH stimulates both follicular and parafollicular cells in the rat thyroid and that iodine deficiency causes hypercalcemia and hypercalcitonemia.

Interactions of cholesterol and cholesterol sulfate with free fatty acids: possible relevance for the pathogenesis of recessive X-linked ichthyosis.

Whereas the stratum corneum contains large amounts of unesterified cholesterol and minimal amounts of cholesterol sulfate, in recessive X-linked ichthyosis (RXLI), levels of cholesterol decrease while cholesterol sulfate content increases. To study the molecular basis for abnormal shedding in RXLI, we compared the interaction of cholesterol and cholesterol sulfate with the free fatty acid, hexadecanoic acid, by differential scanning calorimetry (DSC). While cholesterol and the free fatty acid formed a eutectic mixture, such an interaction did not occur upon mixing of cholesterol sulfate with hexadecanoic acid. In addition, and unexpectedly, free cholesterol appeared to undergo progressive autoxidation during repeated DSC measurements at only slightly supraphysiologic temperatures. These studies may provide a molecular mechanism for the abnormal desquamation that occurs in RXLI. The regular formation of oxidation products of cholesterol observed here, if matched by equivalent molecular events in vivo, may have important implications for epidermal pathophysiology.

Penetration of dimyristoylphosphatidylcholine monolayers and bilayers by model beta-blocker agents of varying lipophilicity.

Penetration of model beta-blockers, propranolol, oxprenolol, metaprolol, and nadolol, into dimyristoylphosphatidylcholine (DMPC) monolayers cast on a pH 7.4 phosphate buffer (mu = 0.155 adjusted with NaCl) at 25 degreesC was monitored using a film balance equipped with a Wilhelmy plate for measuring changes in surface pressure. Drug solution (pH 7.4) is injected below the surface of the monolayer. The difference in surface pressure, Delta pi, for each drug concentration added to the monolayer was measured at equilibrium. Delta pi increased with increasing drug concentration. Consistent with the relative lipophilicities, the Delta pi vs drug concentration slopes were as follows: propranolol > metaprolol > oxprenolol > nadolol. The intrinsic surface activity of the beta-blockers was also determined in the absence of the lipid. Differential scanning calorimetry (DSC) measurements were also made on DMPC bilayers in the above buffer. DMPC suspended in buffered drug solutions were scanned over a temperature range of 5 degrees to 40 degreesC at a scan rate of 0.091 degreesC/min. The DSC studies indicate that the DMPC thermotropic phase behavior is modulated by these compounds as follows: propranolol >> metaprolol congruent with oxprenolol > nadolol which agrees with reported partition coefficients as well as the above Delta pi observations. However, an accounting of the intrinsic surface activity of these compounds results in a lower than expected affinity for the DMPC monolayer.

Effect of pH and NaCl on measurements of ionized calcium in matrices of serum and human albumin with a new calcium-selective electrode.

A neutral ligand ion-exchanger type of ion-selective electrode is used in the current version of the Orion Model SS-20 calcium analyzer. We evaluated it over the pH range 4.0 to 8.5, using albumin solutions containing 150 mmol/L NaCl and pooled patients' serum. Within this pH range response was nernstian, and the relationship between potential (in millivolts) and log [Ca2+] at each pH was linear. Variation in Ca2+ with increasing ionic strength (because of added NaCl) was not significantly different between standards and albumin solutions; it decreased in both. In contrast, the proportion of calcium in serum increased with increasing NaCl concentrations. Ca2+ was not bound in whole serum albumin at a pH less than about 3.9. We observed a substantial difference between the degree of Ca2+ binding in solutions of fatty-acid-free human albumin and serum.

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.

Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical, and functional considerations.

In terrestrial mammals, stratum corneum lipids derive from two sources: deposition of lamellar body lipids in stratum corneum interstices and excretion of sebaceous lipids onto the skin surface, resulting in a two-compartment ("bricks and mortar") system of lipid-depleted cells surrounded by lipid-enriched intercellular spaces. In contrast, intracellular lipid droplets, normally not present in the epidermis of terrestrial mammals, are prominent in avian and marine mammal epidermis (cetaceans, manatees). We compared the transepidermal water loss, ultrastructure, and lipid biochemistry of the viable epidermis and stratum corneum of pigeon apterium, fledgling (featherless) zebra finches, painted storks, cetaceans, and manatees to those of humans and mice. Marine mammals possess an even more extensive lamellar-body secretory system than do terrestrial mammals; and lamellar-body contents, as in terrestrials, are secreted into the stratum corneum interstices. In cetaceans, however, glycolipids, but not ceramides, persist into the stratum corneum; whereas in manatees, glycolipids are replaced by ceramides, as in terrestrial mammals. Acylglucosylceramides, thought to be critical for lamellar-body deposition and barrier function in terrestrial mammals, are present in manatees but virtually absent in cetaceans, a finding that indicates that they are not obligate constituents of lamellar-body-derived membrane structures. Moreover, cetaceans do not elaborate the very long-chain, saturated N-acyl fatty acids that abound in terrestrial mammalian acylglucosylceramides. Furthermore, cold-water marine mammals generate large, intracellular neutral lipid droplets not found in terrestrial and warm-water marine mammals; these lipid droplets persist into the stratum corneum, suggesting thermogenesis, flotation, and/or cryoprotectant functions. Avians generate distinctive multigranular bodies that may be secreted into the intercellular spaces under xerotic conditions, as in zebra fledglings; ordinarily, however, the internal lamellae and limiting membranes deteriorate, generating intracellular neutral lipid droplets. The sphingolipid composition of avian stratum corneum is intermediate between terrestrials and cetaceans (approximately equal to 50% glycolipids), with triglycerides present in abundance. In the midstratum corneum of avians, neutral lipid droplets are released into the interstices, forming a large extracellular, lipid-enriched compartment, surrounding wafer-thin corneocytes, with a paucity of both lipid and keratin ("plates-and-mortar" rather than the "bricks-and-mortar" of mammals).(ABSTRACT TRUNCATED AT 400 WORDS)

Neutral lipid storage disease with ichthyosis: lipid content and metabolism of fibroblasts.

Neutral lipid storage disease with ichthyosis is a newly recognized heritable disorder characterized by widespread cellular triglyceride storage. Lipid metabolism in fibroblasts cultured from three affected family members was studied. The stored lipid is triglyceride composed of an unremarkable fatty acid profile and derived from both exogenously-supplied and endogenously-synthesized fatty acids. Lipid storage could not be corrected by prolonged culture in lipid-depleted media. Acetyl CoA carboxylase activity and beta-oxidation of palmitate were both normal. Taken together, these studies exclude a primary defect of fatty acid uptake, over-synthesis or impaired beta-oxidation. Moreover, triacylglycerol lipase activity of homogenates of fibroblasts from patients with NLSDI examined over the range of pH 3.5-8.5 was normal.