Skin Lipids from Saudi Arabian Birds

Saudi Journal of Biological Sciences (2014) 21, 173–177

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Skin lipids from Saudi Arabian birds

Haseeb A. Khan a,*, Ibrahim A. Arif b, Joseph B. Williams c, Alex M. Champagne c, Mohammad Shobrak d a Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia b Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia c Department of Evolution, Ecology and Organismal Biology, Aronoff Laboratory, Ohio State University, Columbus, USA d Department of Biology, College of Science, Taif University, Taif, Saudi Arabia

Received 1 September 2013; revised 10 September 2013; accepted 15 September 2013 Available online 25 September 2013

KEYWORDS Abstract Skin lipids play an important role in the regulation of cutaneous water loss (CWL). Skin lipids; Earlier studies have shown that Saudi desert birds exhibit a tendency of reduced CWL than Separation; birds from temperate environment due to adaptive changes in composition of their skin lipids. Detection; In this study, we used thin-layer chromatography (TLC) for separation and detection of non- Thin-layer chromatography; polar and polar lipids from the skin of six bird species including sooty gull, brown booby, Saudi birds house sparrow, Arabian waxbill, sand partridge, and laughing dove. The lipids were separated and detected on Silica gel G coated TLC plates and quantified by using densitometric image analysis. Rf values of the non-polar lipids were as follows: cholesterol (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids methyl esters (0.84) and cholesterol ester (0.97). Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) and cholesterol (0.73). The results showed the abundance of fatty acids methyl esters (47.75–60.46%) followed by triacylglycerol (12.69–24.14%). The remaining lipid compositions were as follows: cholesterol (4.09–13.18%), ceramide (2.18–13.27%), and cerebroside (2.53–12.81%). In conclusion, our findings showed that TLC is a simple and sensitive method for the separation and quantification of skin lipids. We also reported a new protocol for lipid extraction using the zirconia beads for efficient disruption of skin tissues. This study will help us better understand the role of skin lipids in adaptive physiology towards adverse climatic conditions. ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

* Corresponding author. Address: Department of Biochemistry College of Science, Bld 5 King Saud University P.O. Box 2455, 1. Introduction Riyadh 11451, Saudi Arabia. Tel.: +966 11 4675859. E-mail addresses: [email protected], [email protected] (H.A. Khan). The outermost layer of the skin, the stratum corneum, is com- Peer review under responsibility of King Saud University. posed of ﬂat, dead cells called corneocytes embedded within a lipid matrix (Bouwstra, 1997), which function collectively as the primary barrier to water vapour diffusion from the animal to the environment, a process known as cutaneous water loss Production and hosting by Elsevier (CWL) (Menon et al., 1992; Simonetti et al., 1995).

1319-562X ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.09.008 174 H.A. Khan et al.

The stratum corneum is composed of corneocytes, flat- 2.2. Extraction of skin lipids tened dead cells embedded in a matrix of lipids, primarily ceramides, cholesterol and free fatty acids (Bouwstra et al., Frozen skin samples were placed in cold phosphate buffered 2003) that are organized in bilayer or lamellae that saline (PBS) and the subcutis portion was scratched off using minimizes water permeation through the skin (Wertz, 2000; a scalpel. About 30 mg of minced skin sample was placed in Bouwstra et al., 2003). a 2 ml vial containing 0.5 mm zirconia beads and ice-cold It has been observed that house sparrows from the desert 0.5 ml methanol with 0.01% butylated hydroxytoluene of Saudi Arabia exhibit 25% lower rates of CWL than tem- (BHT). The vial was placed in the slot of a minibead beater perate-zone American sparrows; this adaptive physiology has and the tissue was homogenized for 1 min at 4200 rpm. The been linked to the higher percentages of both intercellular homogenate was mixed with 1 ml chloroform and the contents and covalently bound cerebrosides in the desert species were shaken overnight. Then 250 ll of 0.15 M ammonium ace- (Gu et al., 2008; Munoz-Garcia and Williams, 2005). Due tate was added and the contents were re-homogenized for to the climate change in Saudi Arabia towards extreme tem- 1 min at 4200 rpm. The upper layer was removed and 1 ml peratures, it is expected that over the long term the desert of the lower layer was transferred to an eppendorf tube. The birds in this country will likely be forced to further reduce samples were evaporated to dryness in a rotary evaporator their evaporative water losses if they are to remain in their at 45 C and reconstituted with 100 ll of chloroform:methanol present habitat (Williams et al., 2012). How these desert (2:1) containing 0.01% BHT. The tubes were centrifuged at birds will resolve the anticipated conflicting demands of 2000 rpm for 2 min before performing the TLC. water shortage and thermoregulation remains unknown and is a subject of further research. 2.3. Thin-layer chromatography Studies across a number of bird species from multiple environments may help us understand how skin lipids are orga- We used commercially available TLC plates (20 20 cm) nized in birds relative to CWL. Desert birds tend to have a · pre-coated with 0.25 mm thick layer of silica gel G (Adsorbo- greater proportion of more polar ceramides with longer fatty sil-Plus 1, Altech, Deerfield, IL, USA). The plates were precon- acid tails (Schaefer and Redelmeier, 1996). Haugen et al. ditioned by running in chloroform:methanol (2:1) until the (2003) compared intercellular lipid composition in eight species solution reached the top. The plates were activated by keeping of larks across an aridity gradient however cerebrosides were them in an oven (110 C) for 30 min. Aliquot (5 ll) of standard not tested in that study. Champagne et al. (2012) have found solution of lipids’ mixture and skin sample homogenates were that birds in arid environments have lower rates of CWL than applied onto the plate and the plate was allowed to dry for birds from mesic environments. These differences in water loss 5 min before developing in the mobile phase. may be explained by differences in the interactions between For separation of nonpolar lipids, the TLC plate was devel- and among intercellular lipids, while a negative correlation be- oped in hexane:ethylether:acetic acid (80:20:2) to the top. For tween cerebrosides and CWL has been noticed (Champagne separation of polar lipids, the TLC plates were placed in chlo- et al., 2012). Thus, considering an important role of skin lipids roform:methanol:water (60:40:5) and run to 10 cm distance in climatic tolerance and adaptive physiology, it is intriguing to from the bottom, followed by chloroform:methanol:acetic acid investigate the composition of skin lipids of Saudi Arabian (190:9:1) and run to 15 cm from the bottom, and finally placed birds. in hexane:ethylether:acetic acid (70:30:1) and run to the top. Thin-layer chromatography (TLC) is a simple and rapid The plate was allowed to dry for about 10 min between the technique that has been used for the analysis of lipids (Macala successive runs and after the final run. The plate was then et al., 1983; Ruiz and Ochoa, 1997; Fewster et al., 1969; sprayed with cupric acetate solution (3% cupric acid + 8% Rawyler and Siegenthaler, 1980). In continuation to our previ- phosphoric acid in distilled water) and placed in an oven at ous work on thin-layer chromatographic analysis of biomole- 180 C for 30 min. The lipid bands appear grey on the white cules (Khan, 2006, 2007; Rathore and Khan, 1987, 1988; background. Rf values were calculated using the formula, Rathore et al., 1988), we have performed TLC separation Rf = distance travelled by the analyte/distance travelled by and detection of non-polar and polar lipid fractions in epider- the mobile phase. mal skin of six bird species from Saudi Arabia. 2.4. Densitometry 2. Materials and methods

2.1. Birds and sample collection The TLC plate was wrapped in a transparent plastic and the coating side was scanned using a scanner. The image was saved in a computer and the intensities of lipid-specific spots were Sea birds (sooty gull and brown booby) were collected from an quantified using Image J software (National Institute of island near Jeddah, Saudi Arabia. Other four birds including Health, USA). house sparrow, Arabian waxbill, sand partridge and laughing dove were collected from the local market. The description 3. Results of birds is given in Table 1. The birds were sacrificed and the feathers of the ventral side were removed. The skin of the ventral region (abdominal area) was thoroughly washed with The images of TLC plates displaying the separations of non- antibacterial soap and specimens of 1.5–2.0 cm2 size were cut polar and polar lipids are shown in Figs. 1 and 2 respectively. and placed in media. The samples were stored at À80 C until The Rf values of non-polar lipids were as follows: cholesterol analyzed. (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids Skin lipids from Saudi Arabian birds 175

Table 1 Description of birds used in this study. SN Zoological name Common name Order Habitat 1 Larus hemprichii Sooty gull (adult) Charadriiformes Sea bird 2 Sula leucogaster Brown booby Suliformes Sea bird 3 Larus hemprichii Sooty gull (young) Charadriiformes Sea bird 4 Passer domesticus House sparrow Passeriformes Urban and rural 5 Estrilda astrild Arabian waxbill Passeriformes Grasslands 6 Ammoperdix heyi Sand partridge Galliformes Dry and hilly 7 Spilopelia senegalensis Laughing dove Columbiformes Dry and semi-desert

Figure 1 TLC separation of non-polar skin lipids. STD, lipid Figure 2 TLC separation of polar skin lipids. STD, lipid standards; 1, sooty gull (adult); 2, brown booby; 3, sooty gull standards; 1, sooty gull (adult); 2, brown booby; 3, sooty gull (young); 4, house sparrow; 5, Arabian waxbill; 6, sand partridge; (young); 4, house sparrow; 5, Arabian waxbill; 6, sand partridge; 7, laughing dove. 7, laughing dove. methyl esters (0.84) and cholesterol ester (0.97). The Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) 4. Discussion and cholesterol (0.73). Cholesterol ester was not detected in the skin of any of the bird samples. Free fatty acids were also Our results showed that TLC is a simple and efficient tech- undetected in all the birds’ skins except the sand partridge nique for separation of different classes of skin lipids with (Fig. 1). There were some unidentified lipid fractions with Rf good resolution. Previous studies have shown that lipids of values different to the lipid standards, on both non-polar the avian stratum corneum consist primarily of cerebrosides, and polar plates. ceramides, cholesterol, free fatty acids, triacylglycerides, fatty The most abundant lipids were fatty acids methyl esters acid methyl esters and cholesterol esters (Haugen et al., (47.75–60.46%) followed by triacylglycerol (12.69–24.14%) 2003; Munoz-Garcia et al., 2006; Ro and Williams, 2010). In (Fig. 3). The complex lipid compositions were as follows: cho- our study, none of the birds’ skin showed the presence of cholesterol (4.09–13.18%), ceramide (2.18–13.27%), and cerebro- lesterol esters and free fatty acids (except sand partridge). This side (2.53–12.81%) (Fig. 3). Among all the species tested, may be due to the use of a small sample size (30 mg tissue) in sand partridge showed the lowest level of triacylglycerol our study that may not be sufficient enough for low abundance (12.69%) and highest level of cholesterol (13.18%). On the lipids to reach the detection limit of TLC. other hand, Arabian waxbill had lowest cholesterol (4.09%) Sphingolipids, including ceramide, are abundantly present and ceramide (2.18%) but highest level of cerebroside in animal epidermis (Lampe et al., 1983) and are major inter- (12.81%). Both adult and young sooty gulls had similar levels cellular lipids that maintain the skin barrier function (Holleran of fatty acids methyl esters, triacylglycerol and cholesterol. et al., 2006; Jennemann et al., 2007). A unique difference be- However, the adult sooty gull had comparatively higher levels tween the mammalian and avian stratum corneum is the pres- of ceramide but lower levels of cerebrosides as compared to the ence of cerebrosides in the latter taxa (Munoz-Garcia and young sooty gull (Fig. 3). Williams, 2005; Gu et al., 2008). In mammalian stratum cor- 176 H.A. Khan et al.

Figure 3 Percent composition of non-polar and polar lipids in skins of different bird species. neum, the enzyme b-glucocerebrosidase cleaves the sugar This is the first study on measuring the composition of skin lip- group from cerebrosides before they reach the stratum cor- ids in Saudi Arabian birds and will help to better understand neum to convert them into ceramides. Whereas, in bird species, the role of skin lipids in adaptive physiology towards adverse b-glucocerebrosidase converts some but not all of the cerebro- climatic conditions. sides into ceramides (Cox et al., 2008), resulting in cerebrosides making up a substantial portion of the lipids in the stratum Acknowledgments corneum (Munoz-Garcia and Williams, 2005; Gu et al., 2008). We observed multiple bands near the Rf values of cera- This study was supported by a grant (No. 10-BIO-1116-02) mides and cerebrosides indicating the existence of similar lipids from the National Plan for Science and Technology (NPST) in birds’ skin (Fig. 2). Champagne et al. (2012) have reported a of King Saud University, Riyadh, Saudi Arabia. The assis- number of ceramide types in the stratum corneum of birds and tance of Ibrahim Saleh Hadi, Ahmad Al Asfar and Ahmad they classified these ceramides into three distinct groups. Mustafa in sample collection is highly appreciated. The quantitative densitometric analysis showed an abundance of fatty acids methyl esters followed by triacylglycerol References whereas the percentages of cholesterol, ceramide, and cerebroside were quite similar (Fig. 3). While computing the percent Bouwstra, J.A., 1997. 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Lampe et al. Cox, R.M., Munoz-Garcia, A., Jurkowitz, M.S., Williams, J.B., 2008. (1983) have shown that phospholipid species comprised about Beta-Glucocerebrosidase activity in the stratum corneum of house 45% of the total lipid in the basal and spinous layers; they sparrows following acclimation to high and low humidity. Physiol. consisted of only about 25% of the total lipid in the stratum Biochem. Zool. 81, 97–105. granulosum and less than 5% of the total lipid in the stratum Fewster, M.E., Burns, B.J., Mead, J.F., 1969. Quantitative densito- corneum. Neutral lipids began to predominate over phospho- metric thin-layer chromatography of lipids using copper acetate lipids in the stratum granulosum where they comprised over reagent. J. 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