Material and methods

Three skin punch biopsies, three skin scrapings and three skin scrubs were taken from the inguinal region and the caudal back from each of five dogs. Lipid fractions were separated by HPTLC and analysed densitometrically. Additionally, identification

of ceramide classes was verified by electrospray ionisation tandem mass spectrometry (ESI-MS/MS).

Animals

The five dogs, which were cases in a university clinic for small animals and euthanized for reasons not related to this study, were of different breeds (German short-hair pointer, Bernese mountain dog, Dalmatian, two mix breeds) with a median age of 11.4 years (range 4.0 - 14.7). The owners agreed to the use of the dogs for research. None of the dogs had a history or clinical signs of skin disorders. No medications that could possibly influence the skin composition, especially corticosteroids, were given systemically for at least 8 weeks prior to euthanasia. Any kind of topical application had to be stopped 2 weeks before sampling. The lack of abnormalities of the skin was confirmed by a histological examination of additionally taken skin punch biopsies.

Skin sampling and sample preparation

Samples were taken within 3 h postmortem. In order to avoid contamination by hairs, the coat of each dog was carefully clipped at the caudal back and at the inguinal region prior to the sampling.

The biopsy punches were 8 mm in diameter (kai Europe GmbH; Solingen, Germany).

All of the subcutaneous fat was removed immediately, and the epidermis was harvested as described by Kligman and Christophers³² and modified by Stahl et al.³³ The skin biopsies were heated on a plate heater at 60°C for 2 min. After removal of the remaining hair, the epidermis was separated from the dermis carefully using a pair of tweezers.

The scrapes were collected with a surgical blade (B. Braun 10; Aesculap AG, Tuttlingen, Germany) from an area of approximately 10 mm x 18 mm. For each sample, the skin was scraped consecutively 30 times.

The skin scrub technique was adapted from a method for collecting cutaneous bacteria in humans³⁴ and dogs.^35,36 For this procedure, a metal cylinder of 21 mm inner diameter was pressed tightly on the skin and filled with 1 mL of a mixture of

n-hexane and ethanol (2:1 v/v). The solution was stirred by means of a roughened glass rod with slight pressure applied for 30 s and transferred into a glass tube. This procedure was done twice for each specimen. All skin samples were frozen at -80°C immediately after collection.

Lipid extraction

Lipids were extracted as previously described by Bligh and Dyer.³⁷ Briefly, the specimens were homogenized in a suspension of methanol, chloroform and distilled water (2:1:0.8 v/v/v). Changing the ratio of the solvents to 2:2:1.8 (v/v/v) with subsequent shaking resulted in phase separation. The upper hydrophilic phase was carefully aspirated and discarded. The lipid containing lower phase was filtered through a syringe filter (Chromafil AO-4513; Macherey-Nagel GmbH & Co. KG, Düren, Germany), dried in a SpeedVac (Concentrator Plus; Eppendorf, Hamburg, Germany) at 60°C and stored at -20°C until analysis. All solvents used were of HPLC grade (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).

Lipid standards

The following lipids were used as standards for the respective lipid fractions:

phosphatidylcholine, phosphatidylethanolamine, sodium cholesteryl sulphate, galactocerebrosides, α-hydroxy fatty acid ceramide (CerAS), nonhydroxy fatty acid ceramide (CerNS), oleic acid and glyceryl trioleat (triolein; all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany), ceramide VI (CerAP), ceramide III (CerNP) and ceramide I (CerEOS; all from Evonik Industries AG, Essen, Germany), cholesterol and cholesteryl stearate (both from Fluka Chemie AG, Buchs, Switzerland). For each standard, the lower limit of detection and standard curves were assessed. The standard curves were not linear but followed a Michaelis-Menten function as previously described.³⁸ The standards were mixed in six increasing concentrations, where standard mix ‘0’ corresponding to the lower detection limit. In order to avoid measurement errors due to the manual character of the method, the six standard solutions were always applied additionally to the specimen extracts on silica gel plates.

High performance thin layer chromatography (HPTLC)

In order to separate all major epidermal lipid fractions simultaneously, we modified the HPTLC procedure developed by Stahl et al.³³ The plates (HPTLC Silica gel 60, 20 cm x 10 cm; Merck KGaA, Darmstadt, Germany) were precleaned in a mixture of chloroform and methanol (1:1 v/v) and activated in an oven (model 600; Memmert GmbH & Co.KG, Schwabach, Germany) at 110°C for 10 min. The extracts were dissolved in 200 µl (heat-separated epidermis and scrapes) or 500 µl (scrubs) of a mixture of chloroform and methanol (1:1 v/v). Ten, five and three microlitres of each extract, as well as 10 µL of each standard solution, were applied using a microlitre syringe (701 N; Hamilton Bonaduz AG, Bonaduz, Switzerland). Lipids were separated consecutively in four different developing chambers (CAMAG Chemie-Erzeugnisse & Adsorptionstechnik AG & Co. GmbH, Berlin, Germany) filled with 50 mL of the following solution systems: (i) chloroform, methanol and acetic acid (80:18:2 v/v/v) to 4 cm; (ii) chloroform, methanol and acetic acid (91.4:4.3:4.3 v/v/v) to 8 cm; (iii) n-hexane, diethylether and acetic acid (72.7:18.2:9.1 v/v/v) to 9.5 cm; (iv) n-hexane to the top. In order to obtain visible bands, the developed plates were dipped in an aqueous solution of copper sulphate (75 g/L; copper (II) sulfate-pentahydrate GR; Merck KGaA) and phosphoric acid (8.5%; phosphoric acid 85%;

Sigma-Aldrich Chemie GmbH) for 5 s followed by charring in an oven at 170°C for 15 min. All solvents used were HPLC grade (Carl Roth GmbH & Co. KG).

Densitometry and quantification

The plates were scanned (Scanjet G3010; Hewlett-Packard Company, Palo Alto, CA, USA) and evaluated using the software program ImageJ (software version 1.43u;

public domain, http://rsbweb.nih.gov/ij). Lipid fractions [phospholipids (PL), phosphatidylethanolamine (PE), cholesterol sulphate (ChSO4), glucosylceramides (GlcCer), ceramides (Cer), cholesterol (Chol), free fatty acids (FFA), triglycerides (TG) and cholesteryl ester (ChE)] were identified by comparison of co-migrated standards (Figure 1) as well as their retardation factors (Rf values; ratio of the migration distance of the analyte to the migration distance of the solvent front) and quantified by calculation of respective standard curves. For the ceramide fractions,

CerAP standard was used for the bands of CerAH and CerAP, CerAS standard for the bands of CerAS+NH and CerEOH, CerNP standard for the band of CerNP, CerNS standard for the band of CerNS, CerEOS standard for the bands of CerEOS and CerEOP. All values were given as micrograms per square centimetre. For further calculations, the median of the triple measurements was assessed. Total lipid amount (Lip) was calculated by summation of all detected lipid fractions and total ceramide amount (Cer) by summation of all measured ceramide classes. To compare the lipid and ceramide profile, relative values of each lipid fraction and each ceramide class were calculated based on the total lipid amount and total ceramides, respectively. To answer the question regarding the influence of different ceramide standards for the quantification of ceramides, the bands of CerAP, CerAS, CerNP and CerNS of 12 samples (on three plates) were quantified by each standard, respectively.

Identification of ceramides by mass spectrometry (MS)

Ceramides in the standard solution and a sample separated by HPTLC were further characterized by ESI-MS/MS using a TLC-MS Interface (CAMAG Berlin, Germany) coupled to the pump of a 1200 Series Binary LC System (Agilent, Waldbronn, Germany) and to a 4000 QTrap triple quadrupole/linear ion trap mass spectrometer (AB SCIEX, Darmstadt, Germany).

Ceramide bands were extracted using a mixture of chloroform and methanol (1:1 v/v) with 5 mmol/L ammonium acetate at a flow rate of 0.1 mL/min. Ceramide subspecies were identified in the positive ion mode by product ion scans as well as by precursor ion scans monitoring precursor ions, which produce the characteristic ceramide fragment ions of mass-to-charge ratio (m/z) 264 and 282 (ceramides with sphingosine and phytosphingosine base) or m/z 280 and 298 (ceramides with 6-hydroxysphingosine base) upon collision-induced dissociation. The m/z value is defined as the ratio of the exact mass of an ion to the number of its elementary charges (small molecules form ions that carry only one elementary charge, with the m/z value of these ions being identical with their mass). The instrument settings for nebulizer gas (Gas 1), turbogas (Gas 2), curtain gas and collision gas were 50 psi,

55 psi, 20 psi and medium, respectively. The interface heater was on, the Turbo V ESI source temperature set at 350 °C and the ionspray voltage adjusted to 5.5 kV.

For all precursor and product ion scans, the values for declustering potential, entrance potential and cell exit potential were 80 V, 10 V, and 10 V, respectively. The collision energies ranged from 35 to 55 V. The resulting masses were compared with masses of ceramides calculated by the summation of theoretically possible fatty acids coupled with C18-sphingoid bases.

Statistics

The absolute values of total lipids and the percentage values of all lipid fractions and ceramide classes were compared between the different sampling methods and body sites. Given that we assumed the influence of the sampling repetition to be random, the values of the sampling repetitions were averaged.

The test for normal distribution of model-residuals was the Kolmogorov-Smirnov test and visual assessment of Q-Q plots. Parameters that showed a right skewed distribution were converted to logarithm values prior to calculation. However, results from logarithm values were retransformed to their original scale for graphical presentation.

In situations with normally distributed data (absolute values of Lip; percentage values of PE, Cer and Chol), a repeated-measures two-way ANOVA was used. The same procedure was used for log-normally distributed data (percentage values of PL, ChSO4, FFA, TG and ChE as well as of CerAS/NH, CerEOH, CerEOP, CerNS and CerEOS). Post hoc multiple pairwise comparisons were calculated in the case of significant global test. An α-adjustment was not used because it resulted only very occasionally in significant results, which seemed inappropriate in relation to the results of the global tests. For the calculation investigating the effect of each sampling method of neither normally nor log-normally distributed data (percentage values of GlcCer, CerAH, CerAP and CerNP), the Friedman test for repeated measurements was performed. Owing to the limited number of animals, a statistical analysis of the effect regarding the body site was not possible for these four variables.

To answer the question regarding the reliability of measurement, the variance components of ‘dog’ and ‘sampling repetitions within dogs’ were assessed, describing the inter- and intra-individual variance, respectively. The intraclass correlation coefficients ρIC (0 ≤ ρIC ≤ 1) were calculated using the following formula:

(probe)

stratified by method and body site (σ²d = variance component ‘dog’ [VC (dog)] and σ²e = variance component ‘measurement repetitions’ [VC (probe)].

Analyses were carried out with the SAS program (version 9.2; SAS Institute, Cary, NC, USA). The linear models were calculated with the programs ‘Mixed’ and

‘Varcomp’. For nonparametric analysis, a Cochran-Mantel-Haenszel statistic (based on rank scores) was calculated with the procedure ‘Frequency’ (Friedman test).

Significance was defined at P ≤ 0.05.