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Saatov Botir Talatovich, Post-graduate student, Department of Dermatology, Republican Specialized Scientific-Practical Medical Center of Dermatology and Venereology, Uzbekistan Public Health Ministry
E-mail: [email protected]
Umerov Oibek Ilyasovich, Junior researcher, Laboratory of Metabolomics, Acad. O. A. Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences
Study on the composition and concentrations of phosphoglycolipids in the skin of healthy subjects and patients with vitiligo
Abstract: Despite considerable progress recently attained in study on the human skin lipids there is still a number of problems in this area to be solved. As the achievements of our study, the findings on changes in concentrations of total phospholipids and their fractions as well as on cerebrosides in the human skin in vitiligo should be emphasized. Keywords: cerebrosides, fractions, lipids, phosphoglycolipids, skin, vitiligo.
Introduction
The skin is the largest and most visible organ of a human body. It is an outer cover of a human being, a border between the body and the environment. The skin is a live tissue with biochemical and physiological processes proceeding constantly and continuously [1, 11-16]. Human skin has a very complex structure set up ofvarious types of chemical substances. The skin protects a human organism resisting any chemical, physical and microbiological effects [1, 11-16; 2, 3-5]. The mammalian skin consists of definite types of cells bound by a mixture of lipids [2, 3-5].
Various lipids are present both in the skin and in the epidermis. Thus, the epidermis contains nearly equimolar ratios of cholesterol, ceramides and free fatty acids (FFA), the most significant elements to form a well-organized structure ofthe skin lipid domain [3, 824-834; 4, 10-13]. Quantitative or qualitative changes in lipid composition by sex, age, season, localization and many other factors are of high significance for permeability and dehydration of the skin, as well as for onset and progression of some skin disease [5, 88-91].
It should be noted that some limitations and problems hamper analytical studies on lipids of the skin. Lacking perfect spectrophotometry properties and being more hydrophobic than lipids of other tissues and organs, the lipids of skin is the complex object for analytic study [2, 3-5]. Lipid composition of the mammalian skin was examined quite sufficiently [4, 10-13; 6, 120-130; 7, 147-151; 8, 710-717]; however most studies aimed at determination of limited types of lipids. There is no information about wide spectrum of lipids in human skin, the data about phospholipids and complex skin lipids in pathology are scarce.
Extreme significance of elucidation of role of lipids in the mechanism of the human skin functioning in the normal conditions and in pathology taken into account, wide scale study on fraction composition of lipids from the skin is a crucial task for chemical and medical science. The work was initiated to comparatively study
composition and concentrations of phospholipids and glycolipids in the skin of healthy subjects and in the skin of patients with vitiligo.
Materials and methods
The skin bioptats from healthy subjects and of the depigmen-tated areas from patients with vitiligo were the objects of the study. In total 47 samples of skin were examined, 17 normal and 30 pathological among them. The procedures of extraction of total lipids from the skin and their purification of non-lipid additives was derived from the method of Folch [9, 497-509] with recommendations of Kates [10, 74] by means of chloroform: methanol mixture (2:1 w/w). The total lipid extract of skin thus obtained was used to determine total phospholipids and their fraction composition, as well as for estimation of cerebrosides.
Determination of phospholipids and their fractions
Quantitatively phospholipids and their fractions were assessed by the content of phosphorus determined after mineralization of lipid samples with subsequent colorimetric determination of inorganic phosphorus by reaction of Vaskovsky. SF-26 spectrophotometer (LOMO, the Russian Federation) was used to perform the procedure at 825 nm. [11, 129-141].
Fraction composition of phospholipids was studied by means of thin layer chromatography at KSK silica gel in the mixture of chloroform: methanol: acetic acid: water (16:4:1:14). When the solvent front was achieved, the 13 x 18cm. chromatographic plates were taken out of the chamber to be dried. Phospholipid fractions were developed in the iodine vapor. Each fraction thus obtained was scraped off, subjected to mineralization with perchloric acid in aluminum blocks at 200 °C. The method ofVaskovsky et al. [11, 129-141] was used to determine inorganic phosphorus thus obtained.
Cerebrosides
Cerebrosides from lipid extract of the skin were obtained by means of a general method for the preparation of cerebrosides by Uz-man [12, 149-155]. The procedure involves the extraction of total lipids from fresh tissue with a boiling mixture of chloroform-methanol
Section 6. Medical science
and the isolation of the cerebrosides by virtue of their property of accumulating at the interphase zone when dilute trichloroacetic acid is added to the lipid extract. Quantification of cerebrosides was based on determination of galactose, their carbohydrate component. Galactose was determined by method of Radin et al. [13, 789-796] in combination with method of Svennerholm [14, 42-53]. The method is based on reaction of anthrone with cerebrosides diluted in the concentrated H3PO4; orcine prepared on the concentrated H2SO4 was subsequently added. Intensity of red color thus obtained was measured on Agilent Cary 60 spectrophotometer (Agilent, USA) at 505 nm. The processing of the data was performed by means of Statistica 6.1 program packet [15, 40-49]. Statistical significance was set at P value < 0.05.
Results and discussion
Study on phospholipids
Phospholipids are known as amphiphilic lipid molecules comprising a structural basis of membranes and playing a significant role in functional activity of cells and the whole organism. Normal functioning of organs and tissues depends on integrity of membrane phospholipids. Any disorders in the composition and concentrations of membrane phospholipids results in shifts of cell functional activity and initiation of pathological processes.
It should be noted that despite sufficient number of publications on phospholipids ofvarious organs and tissues, the data about human skin phospholipids are practically absent.
We have isolated phospholipids from the healthy subjects' skin to study their total content and fractional composition. The findings from our study demonstrated that the content of total phospholipids in the healthy skin was 1324.4 ± 57.9 ^g of lipid phosphorus per 1 g. of dry tissue or 33.1 mg. of a phospholipid per 1 g. of dry tissue (3.3 %). Thin layer chromatography helped identifying 8 fractions, such as lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidyl-ethanolamine, cardiolipin and phosphatidic acid.
Among these fractions phosphatidylcholine makes 37.6 % of total skin phospholipids (12.45 ± 0.5 mg/g of dry tissue) to be
Study on cerebrosides
Cerebrosides or monoglycosylceramides are the essential gly-colipids in human and animal tissues playing a significant role in the processes of an organism's life activities. Cerebrosides mostly occur in nervous tissue but can be seen in other tissues in small quantities [20]. Although total content of cerebrosides in tissue cells is not considerable they arouse interest of researchers, and recently have been the object of intensive study. It is firstly due to their strong effect on functional activity of biomembranes. Participating in formation oflamellar bodies ofupper skin cover monoglycosylceramides are the significant part of skin lipids and essential barrier of water permeability in the skin [20; 21, 89-95]. It should be emphasized that the role and place of cerebrosides in the life activities of organs
the highest (Table 1). Cardiolipin is the lowest one making 2.2 % of total phospholipids (0.73 ± 0.03 mg/g of dry tissue). Among skin phospholipids lysophosphatidylcholine is in insignificant amounts (3.4 % of total phospholipids). Neutral phospholipid fractions, such as phosphatidylcholine, phosphatidylethanol-amine and sphingomyelin make 81.2 % of total phospholipids, while acidic fractions, such as lysophosphatidylcholine, phos-phatidylserine, cardiolipin and phosphatidic acid make 18.8 % (Table 1). Mean ratio of the acidic fractions to the neutral ones in a healthy person's skin is 0.23. It should be noted that fractional composition of skin phospholipids does not differ from the one in other organs and tissues of a human organism; however there is difference between the quantitative content of the fractions [16, 28-32; 17, 64-66; 18, 3-6; 19, 54-57].
We examined phospholipid content of the affected skin in patients with vitiligo. Vitiligo is a widely spread human skin disease characterized with white spots on the skin. Today, intensive growth ofpatients with vitiligo worldwide can be seen; however up to present etiopathogenesis of the disease remained unestablished, and efficient methods for its treatment are absent.
There were no changes in fractional composition of phospholipids in the skin of patients with vitiligo (the same 8 fractions) but the significant changes in the depigmentated areas of skin in patients with vitiligo can be seen in quantitative content of both total phospholipids and their fractions. In the affected areas of skin in patients with vitiligo total phospholipids made 28.9 ± 1.3 mg. per 1 g. of dry tissue (2.89 %), that is, 15 % less than in the healthy persons' skin. Contrary to phospholipids in healthy skin, increase in lysophosphatidylcholine and phosphatidic acid could be seen in the depigmentated areas of skin in patients with vitiligo (Table 1). In addition, in the patients increase in phosphatidylserine, phosphatidylinositol and cardiolipin but significant reduction in the neutral phospholipid fractions, such as phosphatidylcholine, phosphati-dylethanolamine and sphingomyelin was observed. As it can be seen, phospholipid composition of the skin in patients with vitiligo tends to change significantly.
and tissues, and in all human organism remain slightly explored. Participation of cerebrosides in the onset and progression of pathologies has been practically unexplored; their role in pathogeneses of many human disorders, vitiligo included, remains unclear. We have managed to study the content of cerebrosides in the skin of healthy subjects and patients with vitiligo; the depigmentated areas of skin of the patients were examined.
The findings from our study demonstrated that mean quantitative content of cerebrosides is 283.5 ± 15.7 [xg/1g of dry tissue. Cerebrosides have been found to decrease in depigmentated areas of skin in patients with vitiligo making 245.2 ± 14.1 ^g/g oftissue in the average reducing by more than 13.5 %. The findings are the evidence for the fact that cerebrosides can play a significant role in
Table 1. - Phospholipids in the skin of healthy subjects and patients with vitiligo (%)
No. Phospholipidfractions Healthy subjects (n = 12) Patients with vitiligo (n = 32)
1 Lysophosphatidylcholine 3.4 ± 0.1 5.3 ± 0.2*
2 Sphingomyelin 20.6 ± 0.9 18.2 ± 0.8
3 Phosphatidylcholine 37.6 ± 1.5 34.2 ± 1.8
4 Phosphatidylserine 3.3 ± 0.1 4.5 ± 0.2
5 Phosphatidylinositol 6.1 ± 0.3 7.9 ± 0.4
6 Phosphatidylethanolamine 23.0 ± 1.3 20.1 ± 1.2
7 Cardiolipin 2.2 ± 0.1 4.6 ± 0.2*
8 Phosphatidic acid 3.7 ± 0.1 5.2 ± 02*
Note: * — statistically significance difference: p < 0.05.
pathological processes taking place in a human's skin. Thus, in our study both qualitative and quantitative assessment of a human's skin phospholipids and their fractions, as well as these of cerebrosides has been performed. The data can be considered as an advance in study on a human's skin chemical composition considerably improving our knowledge about its lipid composition.
In earlier studies on skin lipids free fatty acids, ceramides and cholesterol were stated to be essential lipid components of a human's skin occurring in approximately equimolar ratios [6, 120-130]. According to Pappinen et al. [3, 824-834], in human skin there is 27 %, 51 % and 21 % of cholesterol, cerebrosides and FFA, respectively. These authors demonstrated that the stratum corneum contains 15.1 % of lipids per dry tissue weight; level of phospholipids was 0.4 % only. This is significantly lower than in our study. In other studies phospholipids make 9 % of a human's epidermis [22, 55-56]. As it can be seen, according to various authors, quantitative content of a human's skin total lipids, including phospholipids, significantly differs. In our study we have managed to establish that phospholipids in a healthy human's skin make 3.3 % of dry tissue weight.
There were some studies demonstrating changes in skin lipids upon pathology. Bouwstra and Gooris [4, 10-13] reported that
ceramides decreased in the skin of patients with psoriasis, FFA in stratum corneum were found to increase in the ichthyotic skin. In our study we have managed to demonstrate decrease in monogly-cosylceramies (by 13.5 %) in the affected areas of skin in patients with vitiligo.
Conclusion
Despite considerable progress recently attained in study on a human's skin lipids there are still a number of problems in this area to be solved. First, no robust information about all classes of lipids is available; second, the information about qualitative assessment and function is debatable; third, there are no data about lipid composition of a human's skin lipids upon skin diseases. In a way, the findings from our study fill the gap. Phospholipid composition and their fractional content in the human skin have been established. We have managed to determine the content of monoglycosylceramides (cerebrosides) in the human skin. As the achievements, the findings on changes in concentrations of total phospholipids and their fractions as well as on cerebrosides in the human skin in vitiligo should be emphasized. All-sided study on other representatives of the human skin lipids is needed to be conducted.
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