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Water Evaporation Rates From a Model of Stratum Corneum Lipids
Water Eviiporationi Rates from a Model of Stratum Corneum Lipids STlG E. FRIBERG'AND IBRAtiIM KAYALl
Received March 24, 1987, from the Department of Chemistry, Clarkson University, Potsdam, NY 73676. December 21, 1988. ~~~
~~
~~~~
Abstract 0 Water evaporation rates were measured from thin samples of a model layered structure of stratum corneum lipids with 32% water. A model with only free fatty acids present gave the lowest evaporation rates, while a model with only oleic acid gave values -50% higher. Using the total lipid spectrum of stratuni corneum gave a structure with an
evaporation rate between those mentioned above. The diffusion coefficients calculated were one to two orders of magnitude higher than those of stratum corneum, reflecting the iinfluence of the proteinous part of the latter structure. A sample with only saturated fatty acids gave extremely high evaporation rates; in fact, they were of the same magnitude as those from an unprotected water surface.
The stratified. squamous cells found during keratinization serve as a highly resistant barrier which protects animals from dessication,1.2a function well illustrated by the fact that removal of the thin stratum corneum layer from the skin increases the water loss by 25 to 45 times.' The appearance of skin depends on its water content and numerous analyses have been concerned with methods of measuring water contenk3-5 as well as levels ofevaporation.Gs Dry scaly skin, either pathological3 or nonpathological such as that induced after adhesive tape stripping,g after solvent extraction,lo or after treatment with surfactant solutions,11 shows enhanced transdermal wetter transport. It is now well established that the cellular junctions, which are commonly responsible for barrier functions,l2 are too sparse in the stratum corneum to serve as water barriers. Instead, the available evidence13.14 indicates the stratum corneum lipids in the epithelial interstices to be responsible for the barrier to water transport. This relation has led t o a n intense interest in the correspondence between individual structure and the state of association of stratum corneum lipids. Numerous publications have dealt with the composition of the stratum corneum lipids,15-21 and a layered structure has been proposed.22 In this structure23 (Figure 11, the polar compounds are packed iin lamellae with the hydrocarbon chains mirroring each other and the polar groups adjacent to the aqueous layers. Our grciup has evaluated this layered structure24 for rnixtures of free fatty acids characteristic of the plasma CELL CYTOPLASM
membrane f a t t y acJd
1: "P
cholesrbrol
glucosylceramide
Figure 1-Diagram
illustrating a possible arrangement of lipids forming intercellular bilayers in the stratum corneum (from ref 23). 0022-3549/89/0800-0639$0 7.00/Cl 0 7989, American Pharmaceutical Association
Accepted for publication
skin. At the pH of skin,25 these are present as mixtures of free fatty acids and ionized soaps. Such mixtures spontaneously form a layered structure with water in the form of a liquid crystal26 or a plastic crystal.27 The low angle X-ray diffraction pattern of this layered structure is consistent with that from separated human stratum corneum,24 a fact that supports the claim that the model reflects the organization of lipids in the stratum corneum. Indirect corroboration of the model was nolens volens given by the results of Imokawa and Hattori.10 They showed that some lipids from human stratum corneum are extracted extremely fast (<30 s in vivo) by acetone, while another group of lipids are extracted more slowly (exposure of >20 min). The group that was extracted fast (triglycerides, waxes, hydrocarbons)'" consisted of those lipids which the results of our model experiments24 indicate should be assigned to the space between the methyl group layers. Those extracted slowly (fatty acids, phosphatidyl choline, cholesterol, ceramides) were penetrated between amphiphiles in o u r model.24 In a layered structure in general, lipids located between the methyl groups are bound only by dispersion forces and hence are more easily extracted; these results are all in agreement with the results of Imokawa and Hattori.1" With the claim that the model reflects the condition of lipids in the stratum corneum to some extent substantiated, we found an investigation into its water transport properties well justified. In this publication, we compare evaporation from thin layers of the model with values of water evaporation from stratum corneum species and discuss specific lipid components which are essential for the barrier to water transport in the model.
Experimental Section Materials-The materials used for the model of the stratum corneum lipids are given in Table 1.28 The materials were all of the highest available purity and were used without further purification. Preparation of Samples and Identification of Structures-Samples of lipids for the model structure containing 32%water by weight were prepared in the following manner. The free fatty acids were mixed together in a glass tube with a constriction, and were then neutralized to 41% using NaOH. This acidsoap mixture with 32% water produced a lamellar liquid crystal. The rest of the lipids shown in Table I were added to this structure. Oxygen sensitive compounds (i.e., phosphatidylethanolamine and ceramide) were handled in a nitrogen atmosphere. All glassware was rinsed with ether after cleaning to ensure no external lipid contamination. This mixture was centrifuged repeatedly for 48 h. Small-angle X-ray diffraction patterns were obtained from a Kiessig low-angle camera from Richard Seifert. Using samples in sealed glass capillaries, Ni-filtered Cu radiation was used and the reflection was determined by a Tennelec position-sensitive detector system (model PSD-1100). Visual observation was made in a polarized light microscope to find the optical patterns and textures of the different samples. Desiccators of 6000-mL volume containing 300 mL of saturated salt solutions in the lower part a t constant temperature (30 "C) were used to obtain the required relative humidities. The following salts with their resulting relative humidities (RH) a t 30 "C29 were used: LiBr, Journal of Pharmaceutical Sciences I 639 Vol. 78, No. 8, August 7989
Table I-Composition of Model Epidermal Lipid' Component Free Fatty Acidb M yristic Linoleioc Oleic Palmitic Palmitoleic Stearic Phosphatidylethanolamine
Cholestetyl sulfate Cholesterol Triolein Oleic acid palmityl ester Squalene Pristane Ceramides
Source Aldrich Sigma Sigma Sigma Sigma Sigma Avanti Polar Lipids Research Plus Aldrich Sigma Sigma Aldrich Aldrich Sigma
Purity, Yo Wt% in Mixture 99.5 99 99 99 99 99 >99 98 98 99 98 98 96 99
17 10 15 55 5 10 5 5 4 17 22 5 5 4 21
a Taken from ref 28. Free fatty acids mixture was neutralized to 41% using NaOH with water content of 32% in order to produce a lamellar structure (this composition is called the "host" in the text); after the addition of the rest of the lipids, water content was adjusted to remain at 32% per weight (this structure is called the "model lipid" in the text).
6.2; MgCl,, 32.4; KBr, 80.3; KNO,, 92.5; and K,SO,, 97.0.
Evaporation Rates-Samples of lipids were spread uniformly in 0.5-mm thick layers on a 1-cm2area on microscopy slides and placed in the desiccators. The weight loss from the samples stored in the desiccators with controlled relative humidity was determined hourly by weighing the samples. The weighing was made in ambient atmosphere. The short time exposure to ambient atmosphere had no influence on the overall rate as shown by identical final weights of samples which remained in the dessicator the entire time of the experiment.
Results Evaporation Rates-The evaporation rates from the samples are shown in Figure 2A-C: Figure 2A shows the weight loss from samples containing 32% water plus the free fatty acid soap combination according to Table I (host); Figure 2B shows the loss from a liquid crystal of oleic acid:sodium oleate plus 32%water; Figure 2C shows the numbers from a sample with the total lipid composition according to Table I plus 32% water. As expected, the loss was highest a t the lowest relative humidity and vice versa for all compositions. The lowest loss was shown by the carboxylic acid combination (the host). Addition of the remaining lipids gave an increase in the evaporation rate. The sodium o1eate:oleic acid combination also gave a higher evaporation rate than the host. Evaporation rates for a combination with only saturated fatty acids were extremely high. They actually were at a similar level as those from a free water surface (Table 11); that is, -10 times the values from other lipid combinations. Microscopy Photographs-The microscopic appearance under polarized light of the sample with the total lipids plus 32% water is shown in Figures 3A-C. Before evaporation (Figure 3A), the pattern of the sample with all acids present is characteristic of a slurry of microcrystals embedded in a lamellar liquid crystal. The pattern in Figure 3B is characteristic of liquid crystals. The sample was held a t 97%relative humidity for 2 d and the weight loss was small. As a contrast, the sample exposed to 6.2% relative humidity (Figure 3C) underwent a pronounced change. The liquid crystal pattern disappeared and was replaced by crystals in the form of long needles. Obviously, a phase separation took place and soap or acid:soap crystals were separated from the liquid crystal. The sample with sodium o1eate:oleic acid and water was entirely liquid crystalline at the beginning, but crystals formed in this sample also when the water content was reduced during 640 1 Journal of Pharmaceutical Sciences Vol. 78,No. 8,August 1989
exposure to an atmosphere of 6.2%humidity. Even unsaturated soaps will separate as crystals from a liquid crystal where the water content is sufficiently low.26 Figure 4A shows crystals embedded in the liquid crystals after storage of the sodium o1eate:oleic acid:water sample for 2 d at 32%relative humidity, while the corresponding exposure to 6.2% relative humidity caused a great number of crystals to form (Figure 4B).
Discussion The results provide information on several topics of interest in the analysis of the function of stratum corneum as a barrier to water transport through the skin. In the first instance, the evaporation rates (Figures 2A-C) showed a strong dependence on relative humidity. Figure 5 compares the form of the evaporation rate curves from the total lipid model with the flux of water through stratum corneum in viv0,30 and shows both the evaporation rates during the first and second hour of the model and, as a comparison, i n viv030 values multiplied by 10. The form of the curves is strikingly similar. The difference in magnitude should be viewed as a consequence of the fact that the model consists of lipids only, while the in vivo transport rate is influenced by the presence of tenatinous bodies. Secondly, the values in Figures 2A-C were used to calculate the diffusion coefficient for water through the structure. Following Cooper and Berner,31 the release of material in one direction is calculated. The equation describing the release of material from a membrane having thickness I, is given by+
where M , is the amount of material diffused out of the membrane a t the time t, and M, is the amount of material originally present in the membrane. Realizing that eqs 2 and 3 hold, the consequences shown in eqs 4 and 5 are obvious:
ierfcx
=
1
-
xerfcx
7l
(4)
(5)
Plotting MJMDversus tl" gave the values in Table I11for the diffusion coefficient from the slope of initial rates. A comparison with the diffusion coefficient for the skin30 shows values to two orders of magnitude higher in the present investigation. This is as expected: the water transport through stratum corneum also depends on the corneocyts, the lipid part constitutes only a small fraction of it.33 The influence on the diffusion by the corneocytes cannot be exactly estimated, but it is obvious that the high molecular weight of the proteins and the fact that they are stationary means a strong reduction of the diffusion in their presence.34 The fact that reaggregated delipidized cells with <1% lipid added showed extremely high water transport14 is not a valid
T
A Host
RH o
6.2
0
80.3
*
92.5
32.4
L
00
:3
A
Oleic actd:Oleat
'4
RH 0
97.0
6
LI
80.3
*
92.5 97.0
6
b
Timehours)
A
6.2
32.4
Time(hours) C Model lipid
10 RH 0
n L
$
0
4
\
E"
6.2 32.4 80.3 92.5 97.0
Y
v) v)
0 -1
b 5
c
I .
OO
6. Time(hours1 Figure 2-(A) Evaporation rate of a layered structure of t h e fattyacids according to Table I , saponified to 41% and with 32% water. (B) Evaporation rate of a layered structure ot oleic acid according to Table I, saponified to 41% and with 32% water. (C) Evaporation rate of a layered structure of the lipids according to Table I , with the fatty acids saponified to 41% and with 32% water. argument for a rapid transport of water through the corneocytes. In that case, the rapid transport is due to direct flux through open pores between the cells and the results give no information about the water diffusion through stratum corneum protein components. The transport through an intact complete stratum corneum layer should rather be depicted as taking place both through the lipid layer combined with a flux through a chromatographic matrix of protein, the corneocytes. With this assumption, our values are at a reasonable level between the self diffusion of water in water35 and the diffusion of water through separated stratum corneum.30 The fact that the value of the diffusion coefficient increased with the water
3
content (Table 111) is in accordance with the results on stratum corneum."o The most striking result is the fact that a model of the partially saponified free fatty acids gave a slightly lower diffusion coefficient than the model containing all the lipids of the stratum corneum. A reasonable conclusion appears to be that the decisive element in the stratum corneum resistance to water transport is the fatty acid soap structure. This statement is in agreement with the fact that early attempts to assess the water barrier of specific lipids36 failed. In these cases, organic solvents were used to extract lipids from stratum corneum and the layered structure presumably was perturbed in a nonspecific manner. Rather, the present Journal of Pharmaceutical Sciences i 641 Vol. 78, No. 8, August 1989
A
Table ICRate of Evaporation for Saturated Free Fatty Acid Compared with That of Unprotected Water Layer at 30 "C Relative Humidity 6.2 32.4 80.3 92.5 97.0
Rate of Evaporation, mglcm'ih Saturated Fatty Acids
Pure Water
38.6 30.6 13.0 8.6 3.2
49.0 39.0 16.0 10.0
3.5
A
B
B
Figure &The microscopy patterns in polarized light of the oleic acid: oleate after 2 d at 32.4% relative humidity (A) and at 6.2% humidity (B).
C
Relative Humidity (96)
Figure !&A comparison of evaporation rates from the model of stratum corneum during the first (0)and the second (A)hour, with in vivo results (---) taken from ref 28; the tatter values are multiplied by 10.
Figure +The microscopy patterns in polarized light of the model lipid after preparation (A), after 2 d at 97% relative humidity (B), and at 6.2% relative humidity (C).
results indicate the layered structure as such to be primarily responsible for the barrier, while the presence of individual lipids plays only an indirect role. 642 I Journal of Pharmaceutical Sciences Vol. 78, No. 8, August 1989
Another feature is the fact that a model of the oleic acid: sodium oleate combination gave a higher diffusion coefficient than the total acid combination, while saturated fatty acids obviously gave no resistance to water transport. The values of the latter were close to those of the evaporation from an unprotected water surface (e.g., with no resistance to water transport). The reduction of transport in the presence of saturated
Table Ill-Diffuslon Coefflcient for Water in Layered Models for Stratum Corneulri Lipids"
Relative
"Host"
"Model Lipid"
Humidity
FFAb
(Table I)
Oleic Acid:Oleate
6.2 32.4 80.3 92.5 97.0
5.9 7.1 8.1 11.5 9.7
8.3 8.9 15.7 24.7 28.0
8.7 1 1 .o 12.2 20.8 15.7
a
Units are cm2/s x 1 O-'.
Free fatty acid.
hydrocarbon chains in a crystalline packing was well established two decades ago in the extensive investigations on the prevention of evaporation from stagnant water surfaces37 by monomolecular layers. The rigidity of the saturated chain does not allow water molecules to find pathways through the hydrocarbon la.yer the same way as in a n environment of unsaturated chains. Althouglh evidence is still missing, it is tempting to assign the reduced diffusion coefficient in the presence of saturated fatty acids to an increased rigidity ofthe hydrocarbon chains. The extremely high passage through the model of stcaric acid:sodium stearate is understood also by reference to the evaporation experiments. A monomolecular layer of saturated hydrocarbon37 showed increased resistance towards water evaporation with reduced area per molecule in surface balance determinations, provided the collapse point of the monomolecular layer was not reached. Compression beyond the collapse point led to water evaporation rates in excess of those from a ]pure water ~urface.37The reason for this phenomenon is presumably identical to the reason for the high water trainsport rate through the stearic acid:sodium stearate model in the present investigation. The saturated fatty acid chairis are crystallinely packed a t 30 "C, but the sample does not form a single crystal. Dislocation lines, as well as macroscopic fractures, are frequent in the structure. Cracks exposing the polar groups result in a huge surface area and evaporation is strongly enhanced. These cracks are a serious impediment in the resistance to water transport, and the information about the condition of skin in animals which have been exposed to a diet without these fatty acids is a n excellent illustration of the influence of liquid crystalline hydrocarbon chains. Animals on this diet show both a dry scaly skin and enhanced water evaporation.39 These results give further sulpport that our model reflects the state of the stratum corneum lipids in vivo. However, when judging the final applicability of the model, caution must be exercised. Aside from the complexity of the stratum corneum with the influence of the keratinic compounds, the investigations so far have left no explanation of the problem of the specificity of linoleic acid for the permeability barrier t o water.40
3. Johnson, C.; Shuster, S. J . Invest. Dermatol. 1969, Suppl. 4, 81, 40. 4. Leveque, J. L.; Carson, J. C.; de Rigal, J. J . SOC. Cosmet. Chem. 1979,30, 333. 5. Sedin, G.; Hammarlund, K.; Stromberg, B. Acta Pediatr. Scan. 1983, Suppl. 305,27. 6. Spruit, D.; Malten, K. E. Dermatologica 1969,138, 418. 7. Grice, K.; Sattor, H.; Baker, H. J . Invest. Dermatol. 1972.58.343. 8. Hammarlund, K.; Nilsson, G. E.; Oberg, P. A,; Sedin,'G. 'Acta Pediatr. Scand. 1977, 66, 553. 9. Tagani, H.; Yoshikuni, K. Arch. Dermatol. 1985, 12, 542. 10. Imokawa, G.; Hattori, M. J . Invest. Dermatol. 1985, 84, 282. 11. Fulmer, A. W.; Kramer, G. J. J . Invest. Dermatol. 1986,86, 598. 12. Stoelienlin, L. A. Int. Rev. Cylol. 1974, 39, 191. 13. Elias, P. M.; McNutt, N. S.; Friend, D. S. Anat. Rec. 1977, 189, 577. 14. Smith, W. P.; Christenson, M. S.; Nachet, S.; Gans, E. H. J . Invest. Dermatol. 1982, 78, 7. 15. Elias, P. M.; Goerke, J.; Friend, D. S. J . Invest. Dermatol. 1977, 69, 535. 16. Gray, G. M.; White, R. J. J . Invest. Dermatol. 1978, 70, 336. 17. Gray, G. M.; Yardley, H. J. J . Lipid Res. 1975, 16, 435. 18. Bligh, E. G.; Dyer, W. J. Can. J . Biochem. Physiol. 1959,37,911. 19. Abraham, W.; Wertz, P. W.; Downing, D. T. J . Lipid Res. 1985, 26, 761. 20. Ranasinghe, A. W.; Wertz, P. W.; Downing, D. T.; Mackenzie, J. C. J . Invest. Dermatol. 1986, 86, 187. 21. Elias, P. M. J . Invest. Dermatol. 1983, 80, 44. 22. Elias, P. M.; Brown, B. E.; Fritsch, P. 0.; Lzoeke, R. J.; White, G. M.; White, R. J. J . Invest. Dermatol. 1979, 73, 339. 23. Elias, P. M. J . Invest. Dermatol. 1981,20, 1. 24. Friberg, S. E.; Osborne, D. W. J . Disp. Sci. Technol. 1985, 6(4), 485. 25. Abe, T.; Mayuzumi, J.;Kikuchi, N.; Arai, S. Chem. Pharm. Bull. 1980,28(2), 387. 26. Ekwall, P. In Advances i n Liquid Crystals; Brown, G.H., Ed.; Academic: New York, 1975; p 1. 27. L. Goldsmith, in preparation. 28. Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M. J . Lipid Res. 1983,24, 120. 29. Nyqvist, H. Znt. J . Pharm. Technol. Prod. Mfr. 1983, 4(2), 47. 30. Blank, I. H.; Maloney, J.; Emslie, A. G.; Simon, J.; Apt, C. J . Invest. Dermatol. 1984, 82, 188. 31. Cooper, E. R.; Berner, B. In Methods in Skin Research; Skerron, D.; Skerron, C. J.,Eds.; John Wiley: New York, 1985; Chapter 15. 32. Crank, J.; Park, G. S. Methods of Measurements in Di usion in Polymers; Crank, J.; Park, G. S., Eds.; Academic: Lon on, 1969; pp 15-25. .. 33. Matoltsy, A. G.; Downes, A.M.; Sweeney, T. M. J . Invest. Dermatol. 1968,50, 19. 34. Dick, D. A. T. J . Theor. Biol. 1964, 7, 504. 35. Wang, J. H.; Amundsen, C. B.; Polestra, F. M. J . Am. Chem. SOC. 1954, 76, 4763. 36. Sweeney, T. M.; Downing, D. T. J . Inuest. Dermatol. 1970, 55, 135. 37. Retardation of Evaporation by Monolayers: Transport Processes; LaMer, V. K., Ed.; Academic: New York, 1962. 38. Prottey, C. J . Invest. Dermatol. 1977, 97, 29. 39. Bowser, P. A.; Nugteren, D. H.; White, R. J.; Hantsmuller, U. M. T.; Prottey, C. Biochim. Biophys. Acta 1985, 834, 419. 40. Elias, P. M.; Brown, B. E.; Zioboth, V. A. J. Invest. Dermatol. 1981, 74, 230.
t-
References and Notes 1. Kligman, A. M. In The Epidermis; Montagna, W., Ed.; Academic: New York, 1964; Chapter 20. 2. Elias, P. M.; Friend, D. S. J . Cell Biol. 1975; 65, 185.
Acknowledgments The comments by the reviewers have been helpful.
Journal of Pharmaceutical Sciences I 643 Vol. 78, No. 8, August 1989
Received March 24, 1987, from the Department of Chemistry, Clarkson University, Potsdam, NY 73676. December 21, 1988. ~~~
~~
~~~~
Abstract 0 Water evaporation rates were measured from thin samples of a model layered structure of stratum corneum lipids with 32% water. A model with only free fatty acids present gave the lowest evaporation rates, while a model with only oleic acid gave values -50% higher. Using the total lipid spectrum of stratuni corneum gave a structure with an
evaporation rate between those mentioned above. The diffusion coefficients calculated were one to two orders of magnitude higher than those of stratum corneum, reflecting the iinfluence of the proteinous part of the latter structure. A sample with only saturated fatty acids gave extremely high evaporation rates; in fact, they were of the same magnitude as those from an unprotected water surface.
The stratified. squamous cells found during keratinization serve as a highly resistant barrier which protects animals from dessication,1.2a function well illustrated by the fact that removal of the thin stratum corneum layer from the skin increases the water loss by 25 to 45 times.' The appearance of skin depends on its water content and numerous analyses have been concerned with methods of measuring water contenk3-5 as well as levels ofevaporation.Gs Dry scaly skin, either pathological3 or nonpathological such as that induced after adhesive tape stripping,g after solvent extraction,lo or after treatment with surfactant solutions,11 shows enhanced transdermal wetter transport. It is now well established that the cellular junctions, which are commonly responsible for barrier functions,l2 are too sparse in the stratum corneum to serve as water barriers. Instead, the available evidence13.14 indicates the stratum corneum lipids in the epithelial interstices to be responsible for the barrier to water transport. This relation has led t o a n intense interest in the correspondence between individual structure and the state of association of stratum corneum lipids. Numerous publications have dealt with the composition of the stratum corneum lipids,15-21 and a layered structure has been proposed.22 In this structure23 (Figure 11, the polar compounds are packed iin lamellae with the hydrocarbon chains mirroring each other and the polar groups adjacent to the aqueous layers. Our grciup has evaluated this layered structure24 for rnixtures of free fatty acids characteristic of the plasma CELL CYTOPLASM
membrane f a t t y acJd
1: "P
cholesrbrol
glucosylceramide
Figure 1-Diagram
illustrating a possible arrangement of lipids forming intercellular bilayers in the stratum corneum (from ref 23). 0022-3549/89/0800-0639$0 7.00/Cl 0 7989, American Pharmaceutical Association
Accepted for publication
skin. At the pH of skin,25 these are present as mixtures of free fatty acids and ionized soaps. Such mixtures spontaneously form a layered structure with water in the form of a liquid crystal26 or a plastic crystal.27 The low angle X-ray diffraction pattern of this layered structure is consistent with that from separated human stratum corneum,24 a fact that supports the claim that the model reflects the organization of lipids in the stratum corneum. Indirect corroboration of the model was nolens volens given by the results of Imokawa and Hattori.10 They showed that some lipids from human stratum corneum are extracted extremely fast (<30 s in vivo) by acetone, while another group of lipids are extracted more slowly (exposure of >20 min). The group that was extracted fast (triglycerides, waxes, hydrocarbons)'" consisted of those lipids which the results of our model experiments24 indicate should be assigned to the space between the methyl group layers. Those extracted slowly (fatty acids, phosphatidyl choline, cholesterol, ceramides) were penetrated between amphiphiles in o u r model.24 In a layered structure in general, lipids located between the methyl groups are bound only by dispersion forces and hence are more easily extracted; these results are all in agreement with the results of Imokawa and Hattori.1" With the claim that the model reflects the condition of lipids in the stratum corneum to some extent substantiated, we found an investigation into its water transport properties well justified. In this publication, we compare evaporation from thin layers of the model with values of water evaporation from stratum corneum species and discuss specific lipid components which are essential for the barrier to water transport in the model.
Experimental Section Materials-The materials used for the model of the stratum corneum lipids are given in Table 1.28 The materials were all of the highest available purity and were used without further purification. Preparation of Samples and Identification of Structures-Samples of lipids for the model structure containing 32%water by weight were prepared in the following manner. The free fatty acids were mixed together in a glass tube with a constriction, and were then neutralized to 41% using NaOH. This acidsoap mixture with 32% water produced a lamellar liquid crystal. The rest of the lipids shown in Table I were added to this structure. Oxygen sensitive compounds (i.e., phosphatidylethanolamine and ceramide) were handled in a nitrogen atmosphere. All glassware was rinsed with ether after cleaning to ensure no external lipid contamination. This mixture was centrifuged repeatedly for 48 h. Small-angle X-ray diffraction patterns were obtained from a Kiessig low-angle camera from Richard Seifert. Using samples in sealed glass capillaries, Ni-filtered Cu radiation was used and the reflection was determined by a Tennelec position-sensitive detector system (model PSD-1100). Visual observation was made in a polarized light microscope to find the optical patterns and textures of the different samples. Desiccators of 6000-mL volume containing 300 mL of saturated salt solutions in the lower part a t constant temperature (30 "C) were used to obtain the required relative humidities. The following salts with their resulting relative humidities (RH) a t 30 "C29 were used: LiBr, Journal of Pharmaceutical Sciences I 639 Vol. 78, No. 8, August 7989
Table I-Composition of Model Epidermal Lipid' Component Free Fatty Acidb M yristic Linoleioc Oleic Palmitic Palmitoleic Stearic Phosphatidylethanolamine
Cholestetyl sulfate Cholesterol Triolein Oleic acid palmityl ester Squalene Pristane Ceramides
Source Aldrich Sigma Sigma Sigma Sigma Sigma Avanti Polar Lipids Research Plus Aldrich Sigma Sigma Aldrich Aldrich Sigma
Purity, Yo Wt% in Mixture 99.5 99 99 99 99 99 >99 98 98 99 98 98 96 99
17 10 15 55 5 10 5 5 4 17 22 5 5 4 21
a Taken from ref 28. Free fatty acids mixture was neutralized to 41% using NaOH with water content of 32% in order to produce a lamellar structure (this composition is called the "host" in the text); after the addition of the rest of the lipids, water content was adjusted to remain at 32% per weight (this structure is called the "model lipid" in the text).
6.2; MgCl,, 32.4; KBr, 80.3; KNO,, 92.5; and K,SO,, 97.0.
Evaporation Rates-Samples of lipids were spread uniformly in 0.5-mm thick layers on a 1-cm2area on microscopy slides and placed in the desiccators. The weight loss from the samples stored in the desiccators with controlled relative humidity was determined hourly by weighing the samples. The weighing was made in ambient atmosphere. The short time exposure to ambient atmosphere had no influence on the overall rate as shown by identical final weights of samples which remained in the dessicator the entire time of the experiment.
Results Evaporation Rates-The evaporation rates from the samples are shown in Figure 2A-C: Figure 2A shows the weight loss from samples containing 32% water plus the free fatty acid soap combination according to Table I (host); Figure 2B shows the loss from a liquid crystal of oleic acid:sodium oleate plus 32%water; Figure 2C shows the numbers from a sample with the total lipid composition according to Table I plus 32% water. As expected, the loss was highest a t the lowest relative humidity and vice versa for all compositions. The lowest loss was shown by the carboxylic acid combination (the host). Addition of the remaining lipids gave an increase in the evaporation rate. The sodium o1eate:oleic acid combination also gave a higher evaporation rate than the host. Evaporation rates for a combination with only saturated fatty acids were extremely high. They actually were at a similar level as those from a free water surface (Table 11); that is, -10 times the values from other lipid combinations. Microscopy Photographs-The microscopic appearance under polarized light of the sample with the total lipids plus 32% water is shown in Figures 3A-C. Before evaporation (Figure 3A), the pattern of the sample with all acids present is characteristic of a slurry of microcrystals embedded in a lamellar liquid crystal. The pattern in Figure 3B is characteristic of liquid crystals. The sample was held a t 97%relative humidity for 2 d and the weight loss was small. As a contrast, the sample exposed to 6.2% relative humidity (Figure 3C) underwent a pronounced change. The liquid crystal pattern disappeared and was replaced by crystals in the form of long needles. Obviously, a phase separation took place and soap or acid:soap crystals were separated from the liquid crystal. The sample with sodium o1eate:oleic acid and water was entirely liquid crystalline at the beginning, but crystals formed in this sample also when the water content was reduced during 640 1 Journal of Pharmaceutical Sciences Vol. 78,No. 8,August 1989
exposure to an atmosphere of 6.2%humidity. Even unsaturated soaps will separate as crystals from a liquid crystal where the water content is sufficiently low.26 Figure 4A shows crystals embedded in the liquid crystals after storage of the sodium o1eate:oleic acid:water sample for 2 d at 32%relative humidity, while the corresponding exposure to 6.2% relative humidity caused a great number of crystals to form (Figure 4B).
Discussion The results provide information on several topics of interest in the analysis of the function of stratum corneum as a barrier to water transport through the skin. In the first instance, the evaporation rates (Figures 2A-C) showed a strong dependence on relative humidity. Figure 5 compares the form of the evaporation rate curves from the total lipid model with the flux of water through stratum corneum in viv0,30 and shows both the evaporation rates during the first and second hour of the model and, as a comparison, i n viv030 values multiplied by 10. The form of the curves is strikingly similar. The difference in magnitude should be viewed as a consequence of the fact that the model consists of lipids only, while the in vivo transport rate is influenced by the presence of tenatinous bodies. Secondly, the values in Figures 2A-C were used to calculate the diffusion coefficient for water through the structure. Following Cooper and Berner,31 the release of material in one direction is calculated. The equation describing the release of material from a membrane having thickness I, is given by+
where M , is the amount of material diffused out of the membrane a t the time t, and M, is the amount of material originally present in the membrane. Realizing that eqs 2 and 3 hold, the consequences shown in eqs 4 and 5 are obvious:
ierfcx
=
1
-
xerfcx
7l
(4)
(5)
Plotting MJMDversus tl" gave the values in Table I11for the diffusion coefficient from the slope of initial rates. A comparison with the diffusion coefficient for the skin30 shows values to two orders of magnitude higher in the present investigation. This is as expected: the water transport through stratum corneum also depends on the corneocyts, the lipid part constitutes only a small fraction of it.33 The influence on the diffusion by the corneocytes cannot be exactly estimated, but it is obvious that the high molecular weight of the proteins and the fact that they are stationary means a strong reduction of the diffusion in their presence.34 The fact that reaggregated delipidized cells with <1% lipid added showed extremely high water transport14 is not a valid
T
A Host
RH o
6.2
0
80.3
*
92.5
32.4
L
00
:3
A
Oleic actd:Oleat
'4
RH 0
97.0
6
LI
80.3
*
92.5 97.0
6
b
Timehours)
A
6.2
32.4
Time(hours) C Model lipid
10 RH 0
n L
$
0
4
\
E"
6.2 32.4 80.3 92.5 97.0
Y
v) v)
0 -1
b 5
c
I .
OO
6. Time(hours1 Figure 2-(A) Evaporation rate of a layered structure of t h e fattyacids according to Table I , saponified to 41% and with 32% water. (B) Evaporation rate of a layered structure ot oleic acid according to Table I, saponified to 41% and with 32% water. (C) Evaporation rate of a layered structure of the lipids according to Table I , with the fatty acids saponified to 41% and with 32% water. argument for a rapid transport of water through the corneocytes. In that case, the rapid transport is due to direct flux through open pores between the cells and the results give no information about the water diffusion through stratum corneum protein components. The transport through an intact complete stratum corneum layer should rather be depicted as taking place both through the lipid layer combined with a flux through a chromatographic matrix of protein, the corneocytes. With this assumption, our values are at a reasonable level between the self diffusion of water in water35 and the diffusion of water through separated stratum corneum.30 The fact that the value of the diffusion coefficient increased with the water
3
content (Table 111) is in accordance with the results on stratum corneum."o The most striking result is the fact that a model of the partially saponified free fatty acids gave a slightly lower diffusion coefficient than the model containing all the lipids of the stratum corneum. A reasonable conclusion appears to be that the decisive element in the stratum corneum resistance to water transport is the fatty acid soap structure. This statement is in agreement with the fact that early attempts to assess the water barrier of specific lipids36 failed. In these cases, organic solvents were used to extract lipids from stratum corneum and the layered structure presumably was perturbed in a nonspecific manner. Rather, the present Journal of Pharmaceutical Sciences i 641 Vol. 78, No. 8, August 1989
A
Table ICRate of Evaporation for Saturated Free Fatty Acid Compared with That of Unprotected Water Layer at 30 "C Relative Humidity 6.2 32.4 80.3 92.5 97.0
Rate of Evaporation, mglcm'ih Saturated Fatty Acids
Pure Water
38.6 30.6 13.0 8.6 3.2
49.0 39.0 16.0 10.0
3.5
A
B
B
Figure &The microscopy patterns in polarized light of the oleic acid: oleate after 2 d at 32.4% relative humidity (A) and at 6.2% humidity (B).
C
Relative Humidity (96)
Figure !&A comparison of evaporation rates from the model of stratum corneum during the first (0)and the second (A)hour, with in vivo results (---) taken from ref 28; the tatter values are multiplied by 10.
Figure +The microscopy patterns in polarized light of the model lipid after preparation (A), after 2 d at 97% relative humidity (B), and at 6.2% relative humidity (C).
results indicate the layered structure as such to be primarily responsible for the barrier, while the presence of individual lipids plays only an indirect role. 642 I Journal of Pharmaceutical Sciences Vol. 78, No. 8, August 1989
Another feature is the fact that a model of the oleic acid: sodium oleate combination gave a higher diffusion coefficient than the total acid combination, while saturated fatty acids obviously gave no resistance to water transport. The values of the latter were close to those of the evaporation from an unprotected water surface (e.g., with no resistance to water transport). The reduction of transport in the presence of saturated
Table Ill-Diffuslon Coefflcient for Water in Layered Models for Stratum Corneulri Lipids"
Relative
"Host"
"Model Lipid"
Humidity
FFAb
(Table I)
Oleic Acid:Oleate
6.2 32.4 80.3 92.5 97.0
5.9 7.1 8.1 11.5 9.7
8.3 8.9 15.7 24.7 28.0
8.7 1 1 .o 12.2 20.8 15.7
a
Units are cm2/s x 1 O-'.
Free fatty acid.
hydrocarbon chains in a crystalline packing was well established two decades ago in the extensive investigations on the prevention of evaporation from stagnant water surfaces37 by monomolecular layers. The rigidity of the saturated chain does not allow water molecules to find pathways through the hydrocarbon la.yer the same way as in a n environment of unsaturated chains. Althouglh evidence is still missing, it is tempting to assign the reduced diffusion coefficient in the presence of saturated fatty acids to an increased rigidity ofthe hydrocarbon chains. The extremely high passage through the model of stcaric acid:sodium stearate is understood also by reference to the evaporation experiments. A monomolecular layer of saturated hydrocarbon37 showed increased resistance towards water evaporation with reduced area per molecule in surface balance determinations, provided the collapse point of the monomolecular layer was not reached. Compression beyond the collapse point led to water evaporation rates in excess of those from a ]pure water ~urface.37The reason for this phenomenon is presumably identical to the reason for the high water trainsport rate through the stearic acid:sodium stearate model in the present investigation. The saturated fatty acid chairis are crystallinely packed a t 30 "C, but the sample does not form a single crystal. Dislocation lines, as well as macroscopic fractures, are frequent in the structure. Cracks exposing the polar groups result in a huge surface area and evaporation is strongly enhanced. These cracks are a serious impediment in the resistance to water transport, and the information about the condition of skin in animals which have been exposed to a diet without these fatty acids is a n excellent illustration of the influence of liquid crystalline hydrocarbon chains. Animals on this diet show both a dry scaly skin and enhanced water evaporation.39 These results give further sulpport that our model reflects the state of the stratum corneum lipids in vivo. However, when judging the final applicability of the model, caution must be exercised. Aside from the complexity of the stratum corneum with the influence of the keratinic compounds, the investigations so far have left no explanation of the problem of the specificity of linoleic acid for the permeability barrier t o water.40
3. Johnson, C.; Shuster, S. J . Invest. Dermatol. 1969, Suppl. 4, 81, 40. 4. Leveque, J. L.; Carson, J. C.; de Rigal, J. J . SOC. Cosmet. Chem. 1979,30, 333. 5. Sedin, G.; Hammarlund, K.; Stromberg, B. Acta Pediatr. Scan. 1983, Suppl. 305,27. 6. Spruit, D.; Malten, K. E. Dermatologica 1969,138, 418. 7. Grice, K.; Sattor, H.; Baker, H. J . Invest. Dermatol. 1972.58.343. 8. Hammarlund, K.; Nilsson, G. E.; Oberg, P. A,; Sedin,'G. 'Acta Pediatr. Scand. 1977, 66, 553. 9. Tagani, H.; Yoshikuni, K. Arch. Dermatol. 1985, 12, 542. 10. Imokawa, G.; Hattori, M. J . Invest. Dermatol. 1985, 84, 282. 11. Fulmer, A. W.; Kramer, G. J. J . Invest. Dermatol. 1986,86, 598. 12. Stoelienlin, L. A. Int. Rev. Cylol. 1974, 39, 191. 13. Elias, P. M.; McNutt, N. S.; Friend, D. S. Anat. Rec. 1977, 189, 577. 14. Smith, W. P.; Christenson, M. S.; Nachet, S.; Gans, E. H. J . Invest. Dermatol. 1982, 78, 7. 15. Elias, P. M.; Goerke, J.; Friend, D. S. J . Invest. Dermatol. 1977, 69, 535. 16. Gray, G. M.; White, R. J. J . Invest. Dermatol. 1978, 70, 336. 17. Gray, G. M.; Yardley, H. J. J . Lipid Res. 1975, 16, 435. 18. Bligh, E. G.; Dyer, W. J. Can. J . Biochem. Physiol. 1959,37,911. 19. Abraham, W.; Wertz, P. W.; Downing, D. T. J . Lipid Res. 1985, 26, 761. 20. Ranasinghe, A. W.; Wertz, P. W.; Downing, D. T.; Mackenzie, J. C. J . Invest. Dermatol. 1986, 86, 187. 21. Elias, P. M. J . Invest. Dermatol. 1983, 80, 44. 22. Elias, P. M.; Brown, B. E.; Fritsch, P. 0.; Lzoeke, R. J.; White, G. M.; White, R. J. J . Invest. Dermatol. 1979, 73, 339. 23. Elias, P. M. J . Invest. Dermatol. 1981,20, 1. 24. Friberg, S. E.; Osborne, D. W. J . Disp. Sci. Technol. 1985, 6(4), 485. 25. Abe, T.; Mayuzumi, J.;Kikuchi, N.; Arai, S. Chem. Pharm. Bull. 1980,28(2), 387. 26. Ekwall, P. In Advances i n Liquid Crystals; Brown, G.H., Ed.; Academic: New York, 1975; p 1. 27. L. Goldsmith, in preparation. 28. Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M. J . Lipid Res. 1983,24, 120. 29. Nyqvist, H. Znt. J . Pharm. Technol. Prod. Mfr. 1983, 4(2), 47. 30. Blank, I. H.; Maloney, J.; Emslie, A. G.; Simon, J.; Apt, C. J . Invest. Dermatol. 1984, 82, 188. 31. Cooper, E. R.; Berner, B. In Methods in Skin Research; Skerron, D.; Skerron, C. J.,Eds.; John Wiley: New York, 1985; Chapter 15. 32. Crank, J.; Park, G. S. Methods of Measurements in Di usion in Polymers; Crank, J.; Park, G. S., Eds.; Academic: Lon on, 1969; pp 15-25. .. 33. Matoltsy, A. G.; Downes, A.M.; Sweeney, T. M. J . Invest. Dermatol. 1968,50, 19. 34. Dick, D. A. T. J . Theor. Biol. 1964, 7, 504. 35. Wang, J. H.; Amundsen, C. B.; Polestra, F. M. J . Am. Chem. SOC. 1954, 76, 4763. 36. Sweeney, T. M.; Downing, D. T. J . Inuest. Dermatol. 1970, 55, 135. 37. Retardation of Evaporation by Monolayers: Transport Processes; LaMer, V. K., Ed.; Academic: New York, 1962. 38. Prottey, C. J . Invest. Dermatol. 1977, 97, 29. 39. Bowser, P. A.; Nugteren, D. H.; White, R. J.; Hantsmuller, U. M. T.; Prottey, C. Biochim. Biophys. Acta 1985, 834, 419. 40. Elias, P. M.; Brown, B. E.; Zioboth, V. A. J. Invest. Dermatol. 1981, 74, 230.
t-
References and Notes 1. Kligman, A. M. In The Epidermis; Montagna, W., Ed.; Academic: New York, 1964; Chapter 20. 2. Elias, P. M.; Friend, D. S. J . Cell Biol. 1975; 65, 185.
Acknowledgments The comments by the reviewers have been helpful.
Journal of Pharmaceutical Sciences I 643 Vol. 78, No. 8, August 1989