Chapter 3.5
Skin lipid profiling in normal and seborrhoeic shih tzu dogs
Background – Seborrhoea is a clinical condition resulting in excessive lipid and/or scale on the skin and is a common and important skin disease of dogs. However, there is little information on the skin surface lipid composition of dogs with seborrhoea.
Hypothesis/Objectives – To compare skin surface lipid profiles in normal and seborrhoeic shih tzu dogs.
Methods – Fourteen client-owned dogs (seven seborrhoeic and seven normal) were investigated. Lipids in sebaceous glands (SGs) were extracted from homogenized tissues of SG hyperplasia. Surface lipid was collected by tape stripping [stratum corneum (SC)-enriched fraction] and acetone-wetted cotton swab (acetone-extracted fraction). Lipids in SGs, SC-enriched fractions and acetone-extracted fractions were evaluated by highperformance thin-layer chromatography.
Results – Lipids in SGs mainly consisted of cholesterol esters, wax esters and triglycerides, whereas lipids in the SC-enriched fraction mainly consisted of ceramides. The acetone-extracted fraction contained a mixture of lipid classes recognized in SG- and SC-enriched fractions. In seborrhoeic dogs, concentrations of wax esters and triglycerides in the acetone-extracted fraction were significantly higher than in control dogs (P = 0.0285). Amounts of total ceramides (in micrograms) per milligram of SC were not significantly different between the two groups (P = 0.5204). Interestingly, two unknown ceramide fractions, which accounted for 20% of the total ceramides, were recognized exclusively in seborrhoeic dogs.
Conclusions and clinical importance – These results provide evidence that the skin surface lipid profiles are altered in shih tzu dogs with seborrhoea.
Introduction
In mammals, the sebaceous glands (SGs) and the keratinocytes produce a protective lipid layer that covers the skin. Studies in humans have shown that the lipid composition of SGs differs from that of keratinocytes.1–3 For example, human sebum is primarily comprised of low-polar lipids, such as esters, triglycerides (TGs), diglyceride and squalene, fatty acids and cholesterol (CHOL).1 Meanwhile, human keratinocytes in the stratum granulosum produce ceramides (CERs), CHOL and free fatty acids (FFAs), which comprise the extracellular lipid layers in the stratum corneum (SC) crucial for maintaining the barrier function of the skin.2,3
Seborrhoea is a clinical condition characterized as excessive greasiness and increased scale formation on the skin. This is an important and common skin disease of dogs and it can be secondary to inflammation or be a primary skin disease. In dogs, primary seborrhoea is an inherited disorder of abnormal cornification. Commonly affected dog breeds include the American cocker spaniel, English springer spaniel, West Highland white terrier and Basset hound.4,5 Secondary seborrhoea can result from a wide range of causes, such as ectoparasites, bacterial pyoderma, Malassezia overgrowth, atopic dermatitis, endocrinopathies or nutritional imbalances.4,6 Although primary and secondary seborrhoea are common, very little is known about the composition of skin lipid in this skin disease.
The shih tzu dog breed commonly exhibits seborrhoea, often in association with underlying triggers, such as Malassezia dermatitis or atopic dermatitis.6 The goal of this study was to compare the skin surface lipids of normal and seborrhoeic shih tzu dogs. This information may provide a better understanding of lipid profiles in seborrhoea in dogs.
Material and methods
All dogs were privately owned, and owners gave their written informed consent. All experimental procedures were approved by the Animal Research Committee and were carried out in accordance with the ethical guidelines of Tokyo University of Agriculture and Technology.
Dogs
The normal group of dogs (n = 7) consisted of shih tzu dogs without evidence of skin disease, specifically seborrhoea or Malassezia overgrowth (Table 1). The affected group of dogs (n = 7) consisted of dogs with clinical signs compatible with seborrhoea. The age and gender of the dogs in both groups were matched. Skin cytology revealed Malassezia overgrowth (>10 per high-power field) for all dogs on initial presentation. To minimize the contaminating SC surface lipids with lipids from yeast, all dogs were treated with an antimicrobial shampoo (Malaseb™; Dermcare-Vet Pty Ltd, Springwood, Queensland, Australia) and/or oral itraconazole (5 mg/kg, once daily, Itorizole®; Janssen Pharmaceutical KK, Tokyo, Japan) until the number of Malassezia organisms found on skin cytology were less than two per high-power field (at least 4 weeks). Complete blood counts, serum chemistries, abdominal ultrasound imaging and computer tomography were suggestive of an unilateral adrenal gland tumour in one dog. Three dogs were considered to have atopy, because these dogs had elevated serum allergen-specific IgE. No cause for the Malassezia overgrowth was found in the remaining four seborrhoeic dogs.
Table 1. Signalment of shih tzu dogs at the time of examination
For this study, skin surface lipids and the SC were collected from the seven shih tzu dogs whose clinical conditions were compatible with seborrhoea and the seven normal dogs.
Sebaceous gland tissue
Tissue for SG isolation was collected from two dogs (a 12-year-old male and an 11-year-old spayed female) with SG hyperplasia via skin biopsy procedure using local anaesthesia. Intact hyperplastic SGs were used to maximize collection of pure SG lipids. Skin biopsy specimens were also collected for histological examination; tissue was fixed in 10% neutral buffered formalin and processed routinely.
Lipid extractions
Sebaceous glands.
Lipids were extracted from the SG tissue using a modification of the method reported by Bligh and Dyer.7,8 Briefly, the epithelium was first separated from the SG by incubation with 1500 11/mL dispase II (Godo Syusei, Tokyo, Japan) for 1 h at 37°C. The remaining SG tissue was then homogenized and suspended in a mixture of chloroform, methanol and water (1:2:0.8, by volume) at room temperature for 30 min. Next, chloroform and water were added to the homogenized mixture to change the volume ratio of chloroform, methanol and water to a 1:1:1 mixture. After centrifugation, the lipid-containing supernatant was collected and transferred to new glass test tubes.
Skin surface.
Two methods were used to collect skin surface lipids. In the first, skin surface lipids were collected by vigorously rubbing an area of inguinal skin (2 cm × 4 cm) five times using an approximately 2 cm × 4 cm × 0.5 cm piece of acetone-wetted cotton. This method enabled collection of a mixture of SGs and free extractable SC lipids. The cotton material was then placed into glass tubes, sealed and preserved at -20°C until analysed. For lipid extraction, the cotton swabs were dissolved in 10 mL of a chloroform and methanol mixture (2:1, by volume) at room temperature, and the solvent containing a lipid extract was transferred to a new glass test tube.
In the second procedure, using previously described methods,9 lipids from the SC-enriched fractions were collected via tape stripping. Briefly, after removing sebum by scrubbing a 2 cm × 4 cm area of inguinal skin with acetone using cotton swabs, five consecutive tape strippings were performed. In a previously published study, 10 consecutive tape strips removed almost all layers of the SC in dogs.10 Tape strips were then immersed in n-hexane (Sigma-Aldrich, St Louis, MO, USA) and sonicated to collect the SC extracts. The SC components extracted from the tape strips were filtered through a 0.45 μm DURAPORE-membrane filter (HVHP type; Millipore, Billerica, MA, USA). The SC extracts were weighed, placed in glass test tubes and dissolved in 5 mL of chloroform and methanol (2:1, by volume) at room temperature and shaken for 30 min for lipid extraction. After centrifugation at 1000g for 5 min, the supernatant containing the lipid extracts was transferred to a new glass test tube. Lipid extracts from SG hyperplasia, acetone-containing cotton swabs and tape stripping were dried using a nitrogen stream at 38°C, reconstituted in 50 μL of chloroform and methanol (2:1, by volume), and used for analysis. To avoid technical variance between investigators, all lipid extraction procedures were performed by the same investigator (J.-S.Y.).
High-performance thin-layer chromatography (HPTLC)
Lipid analysis was performed using HPTLC. Briefly, 5 μL reconstituted lipid extract was applied to HPTLC plates (Merck, Darmstadt, Germany). The lipid bands of different polarity were developed using hexane followed by a solvent mixture of chloroform, methanol and acetic acid (190:9:1, by volume). In addition, for the quantitative analysis of ceramide classes, the bands of size-fractionated CER classes were developed by three separate applications of a mixture of chloroform, methanol and acetic acid (190:9:1, by volume). Nonpolar lipid mixtures, bovine-derived CER[NS] (combination of nonhydroxy fatty acids and 6-hydroxyl sphingosines) and CER[AS] (combination of α-hydroxy fatty acids and 6-hydroxyl sphingosines; Matreya, Pleasant Gap, PA, USA) were used as standards to determine the polarity and quantity of each HPTLC band. For colour development, the plates were sprayed with 10% CuSO4 (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 8% H3PO4 (Wako Pure Chemical Industries, Ltd) aqueous solution and then heated at 180°C for 7 min. The HPTLC fractions were scanned and subjected to a density plot analysis using Bio1D-ver.12.11 software (Vilber Lourmat, Marne-la-Vallée, France). The quantity of each CER fraction per milligram of the SC was determined by comparing the density plots with those of serially diluted (0.1, 0.3, 0.6 and 1 μg/μL) CER standards in chloroform and methanol (2:1, by volume). 9
Normal-phase liquid chromatography connected to electrospray ionization-mass spectrometry (NPLC-ESI-MS)
In order to analyse CERs, NPLC-ESI-MS was conducted as previously described.9 Briefly, lipids extracted from SC were redissolved in 10 mL of chloroform and methanol (99.5:0.5, by volume) and applied to Sep-Pak Vac RC Silica cartridges (Waters, Milford, MA, USA). In this method, flow-through fractions contain low-polar lipids (e.g. CHOL), while high-polar lipids (e.g. CERs) appear in the eluate that is extracted from the cartridge using chloroform and methanol (95:5, by volume). The fractions containing CERs were dried using a nitrogen stream and submitted to Yukiguni Aguri Co. (Gunma, Japan) for NPLC-ESI-MS using a LTQ-Orbitrap mass spectrometry system operating in electrospray ionization mode (Thermo Fisher Scientific, Waltham, MA, USA). Parameters for scan measurement of the electrospray ionization using unit mass resolution mode were determined at the following settings: polarity, negative; temperature of nitrogen gas, 300°C; flow of heated dry nitrogen gas, 8.0 L/min; nebulized gas pressure, 137.9 kPa.; capillary voltage, 3500 V; fragmenter voltage, 150 V; and scan range, 250-1500 atomic mass unit.
Statistical analyses
Student’s unpaired t-test (StatView® 5.0; SAS Institute Inc., Cary, NC, USA) was used to compare the relative amounts of lipid fractions, quantities of total CERs, and each CER class per milligram of SC between seborrhoeic dogs and control dogs. A P-value of <0.05 was considered to be statistically significant.
Results
Profiling of skin lipids
Initially, the major lipid components in the SG and surface lipids (i.e. SC-enriched and acetone-extracted fractions) in shih tzu dogs without seborrhoea were determined. Lipids extracted from tissues of SG hyperplasia, SC-enriched fractions and acetone-extracted fractions were subjected to HPTLC analysis. If the polarity of the fractions in canine samples were almost identical to those of the lipid standards, including cholesterol esters (CEs), TGs, CHOL, FFAs and CERs, the fractions were assigned to canine CEs, TGs, CHOLs, FFAs and CERs corresponding to the standard (Figure 1a). A dense band recognized between CEs and TGs was assigned to wax esters (WEs) according to a previous report.11 It was found that lipids extracted from SGs consisted of mainly CEs (18.3%), WEs (19.2%) and TGs (45.2%), as well as small proportions of CHOL (8.5%) and FFAs (5.3%; Figure 1b). In contrast, lipids in the SC-enriched fraction mainly consisted of CERs (65.6%) and small proportions of CHOL (7.7%) and FFAs (11.6%; Figure 1b). Lipids in the acetone-extracted fraction consisted of a mixture of SG- and SC-derived lipids (Figure 1b).
Figure 1. High-performance thin-layer chromatography (HPTLC) analysis of lipids extracted from sebaceous gland (SG) hyperplasia, stratum corneum (SC) and the skin surface in shih tzu dogs without seborrhoea. (a) Representative data from HPTLC analysis of lipids in SG hyperplasia (SG-enriched fraction), SC (SC-enriched tape stripped fraction) and skin surface (acetone-extracted fraction). (b) Composition ratio of lipids in SG hyperplasia, SC and skin surface. Abbreviations: CE, cholesterol ester; CER, ceramide; CER[AS], combination of α-hydroxy fatty acid and sphingosine; CER[NS], combination of nonhydroxy fatty acid and sphingosine; CHOL, cholesterol; FFA, free fatty acid; TG, triglyceride; WE, wax ester.
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