Eight Korean patients with psoriasis (two women, six men) ranging in age from 21 to 58 years gave informed consent and participated in this study. All subjects had psoriasis vulgaris as identified through clinical and histologic assessment and had not been treated either systemically or topically for at least one month before punch biopsies were obtained. Using a 4-mm punch, biopsies were taken from lesional and non-lesional skin on the lower extremities, back, or arms. The epidermis was separated as described previously14. Specifically, the epidermis was separated from whole-skin biopsies by overnight incubation at 4℃ in a 1:1 (v/v) mixture of Dispase solution (Roche Molecular Biochemicals, Manheim, Germany) and Hank's balanced salt solution (HBSS; Gibco BRL, Life Technologies, Rockville, MD, USA).

The clinical severity was assessed using the Psoriasis Area Severity Index (PASI) score, which is calculated as follows: PASI=0.1 (Eh+Ih+Dh) Ah+0.2 (Eu+Iu+Du) Au+0.3 (Et+It+Dt) At+0.4 (El+Il+Dl) Al, where E=erythema, I=infiltration, D=desquamation, A=area, h=head, u=upper extremities, t=trunk, and l=lower extremities. A numerical value is given to the extent of the lesions in each area as follows: 1≤10%, 2=10~30%, 3=30~50%, 4=50~70%, 5=70~90%, and 6=90~100%. E, I, and D are scored on a five-point scale (0=no symptoms, 1=slight, 2=moderate, 3=marked, and 4=very marked) to obtain a final PASI score between 0 and 72. The PASI scores of the patients who took part in this study ranged between 1.8 and 23.7; this range corresponds to mild and moderate psoriasis. Only patients with PASI scores <25 were enrolled in this study in order to determine whether alterations in the levels of ceramides and ceramide-related apoptotic signaling molecules are closely correlated to the clinical severity of mild to moderate psoriasis.

Epidermal tissue isolated from each 4 mm punch biopsy was homogenized with a Polytron homogenizer in 500 µl of ice-cold phosphate-buffered saline. After centrifugation at 240×g for 5 min, aliquots (50 µl of supernatant) of the epidermal homogenates were collected for protein determination by a modified Lowry method using bovine serum albumin as the standard15. Sphingoid bases were extracted by adding 350 µl of MeOH, 150 µl of 1 M NaCl and 300 µl of CHCl₃ to the prepared homogenates. A 35 µg aliquot of 3N NaOH was also added to adjust the pH to 10~11 in order to facilitate the separation of sphingoid bases in the upper phase. As an internal standard, 100 pmol of the non-naturally occurring species15,16 C₁₇ So and C₁₇ S1P was added followed by vigorous vortexing for 1 h. After centrifugation at 240×g for 3 min, the lower phase was evaporated under N₂ gas and then dissolved in 500 µl of 0.15 M methanolic KOH with vortexing for 20 min at 37℃. Then, 500 µl of CHCl₃, 100 µl of 2 N-NH₄OH and 400 µl of alkaline water were added and the mixture was vortexed for an additional hour. After centrifugation at 240×g for 3 min, the lower phase was washed twice with 800 µl of alkaline water and the CHCl₃ was evaporated using a SpeedVac concentrator16.

The extracted lipid fraction was resolved in 120 µl of MeOH. A 20 µl volume of o-phthalaldehyde (OPA) derivatization reagent (Sigma, St. Louis, MO, USA; 50 mg ortho-phthal-dialdehyde, 1 ml ethanol, 100 µl 2-mercaptoethanol, and 50 ml 3% (w/v) boric acid solution) was added and the mixture was allowed to stand for 30 min at room temperature17,18. OPA reacts with the primary amine group of the sphingoid bases and becomes highly fluorescent at an excitation wavelength of 340 nm and an emission wavelength of 455 nm17,18. The derivatives were analyzed using a Jasco (Tokyo, Japan) PU-980 pump, an AS-1559 autosampler, and a Jasco FP-920 fluorescence detector. The isocratic eluent comprised of methanol: deionized distilled water (92:8 v/v) with 0.1% triethylamine was flowed at the rate of 1 ml/min. A 70 µl aliquot of the derivatives was injected then separated on an C₁₈ column (Waters, Sunfire 4.6×1.5 mm internal diameter) kept at room temperature, and the fluorescence was measured at an emission wavelength of 455 nm and an excitation wavelength of 340 nm17. C₁₇ So (8.5 min), C₁₈ So (10.5 min), C₁₈ Sa (14.2 min) were detected.

Isolated epidermis was added to 200 µl of Folch solution (CHCl₃:MeOH, 2:1, v/v mixture) and homogenized using a Polytron homogenizer, and 200 µl of 0.1 M KCl was subsequently added. The mixture was centrifuged at 2,000 rpm for 5 min, and the upper phase containing the extracted proteins was separated. This process was repeated a total of two times. The protein concentration was determined by BCA protein assay (Pierce, Rockford, MD, USA) according to the manufacturer's protocol. Fifty micrograms of protein was fractionated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated with antibodies against ceramide synthase (CerS) and ceramidase (CDase) (Labfrontier, Seoul, Korea) at a 1:250 dilution with Tris-buffered saline containing 0.05% Tween-20 (TSB-T) at 4℃ overnight. After washing with TBS-T for 1 h, the membranes were reacted with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Biosciences, Buckinghamshire, UK) that had been diluted 1:250 with TBS-T, for 1 h at room temperature. After washing the membranes with TBS-T for 2 h, protein expression was detected using an ECL chemiluminescence kit (Amersham Bioscience).

A paired t-test was used to compare the levels of So and Sa between the lesional and non-lesional samples collected from psoriasis patients. The relationship between the CDase levels and PASI scores was assessed using Pearson's correlation analysis. p<0.05 were considered statistically significant.

The high-performance liquid chromatography analysis results of C₁₈ So and C₁₈ Sa in the lesional and non-lesional epidermis samples from psoriasis patients are shown in Fig. 1. The level of So in the lesional epidermis was significantly higher (23.5±10.9; range, 14.66~32.34 nmol/µg protein) than that in the non-lesional epidermis (10.9±4.08; range, 6.82~14.98 nmol/µg protein) (p<0.05). Likewise, the level of Sa in the lesional epidermis was significantly higher (11.4±4.20; range, 7.2~15.6 nmol/µg protein) than that in the non-lesional epidermis (1.3±0.70; range, 0.6~2.06 nmol/µg protein) (p<0.05). The mean % of lesional So to non-lesional So and that of Sa were 215% and 857%, respectively.

The CDase and CerS expression levels in the lesional and non-lesional epidermis of psoriasis patients, PASI scores, and disease durations are listed in Table 1 and 2. The absolute level of CDase and CerS varied from patient to patient. The protein expression of CDase and CerS in the lesional epidermis was not altered compared to that in the non-lesional epidermis. A highly significant positive correlation was observed between the % change in CDase and the PASI score (r=0.752, p<0.05), as shown in Fig. 2, but there was no correlation between the % change in CerS and the PASI score. These results indicate that an increase of CDase is significantly correlated with the PASI score in mild to severe psoriasis, suggesting that increased levels of So are likely caused by the increased expression of CDase.