Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis
2003; Elsevier BV; Volume: 44; Issue: 1 Linguagem: Inglês
10.1194/jlr.m200225-jlr200
ISSN1539-7262
AutoresReiko Okamoto, Junko Arikawa, Mutsumi Ishibashi, Makoto Kawashima, Yutaka Takagi, Genji Imokawa,
Tópico(s)Advancements in Transdermal Drug Delivery
ResumoTo clarify the functional relevance of sphingomyelin (100) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [14C]acetic anhydride to yield [14C-C2]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase.These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD. To clarify the functional relevance of sphingomyelin (100) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [14C]acetic anhydride to yield [14C-C2]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD. Intercellular lipids in the stratum corneum have been implicated as important determinants in the water-retaining properties (1Imokawa G. Hattori M. A possible function of structural lipid in the water-holding properties of the stratum corneum.J. Invest. Dermatol. 1985; 84: 282-284Google Scholar, 2Imokawa G. Kuno H. Kawai M. Stratum corneum lipids serve as a bound-water modulator.J. Invest. Dermatol. 1991; 96: 845-851Abstract Full Text PDF Google Scholar) and in the barrier function (3Holleran W.M. Mao-Qiang M. Gao W.N. Menon G.K. Elias P.M. Feingold K.R. Sphingolipids are required for mammalian epidermal barrier function, inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.J. Clin. Invest. 1991; 88: 1338-1345Google Scholar, 4Imokawa G. Yada Y. Higuchi K. Okuda M. Ohashi Y. Kawamata A. Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid deficient rats and has an inhibitory effect on epidermal hyperplasia.J. Clin. Invest. 1994; 94: 89-96Google Scholar) of the skin. Ceramides are major constituents of intercellular lipids, comprising more than 50% of them. Previously, we demonstrated that there is a marked reduction in the amount of ceramides in the stratum corneum in lesional and in non-lesional forearm skin of patients with atopic dermatitis (AD) (5Imokawa G. Abe A. Jin K. Higaki Y. Kawashima M. Hidano A. Decreased level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin?.J. Invest. Dermatol. 1991; 96: 523-526Google Scholar), which suggested that the ceramide deficiency is an important etiologic factor for the barrier-disrupted and dry skin seen in AD. Subsequently, we found that the causative factor behind the ceramide deficiency in the stratum corneum of patients with AD is an abnormal expression of sphingomyelin (SM) deacylase in their epidermis (6Murata Y. Ogata J. Higaki Y. Kawashima M. Yada Y. Higuchi K. Tsuchiya T. Kawaminami S. Imokawa G. Abnormal expression of sphingomyelin acylase in atopic dermatitis: An etiologic factor for ceramide deficiency?.J. Invest. Dermatol. 1996; 106: 1242-1249Google Scholar). This enzyme hydrolyzes SM at the acyl site to yield free fatty acid and sphingosylphosphorylcholine (SPC) instead of the formation of ceramide and phosphorylcholine (PC) by sphingomyelinase (SMase). Direct enzymatic analysis of the stratum corneum or of the epidermis of patients with AD revealed that there are 9-fold or 3-fold increases, respectively, in the activity of SM deacylase in patients with AD compared with healthy normal controls (7Hara J. Higuchi K. Okamoto R. Kawashima M. Imokawa G. High-expression of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis.J. Invest. Dermatol. 2000; 115: 406-413Google Scholar). The sum of those findings demonstrates that the novel epidermal enzyme SM deacylase is expressed at high levels in the epidermis of patients with AD. The competition of this enzyme with SMase for the common substrate SM leads to the ceramide deficiency in the stratum corneum of patients with AD. Thus in this study, in order to clarify the physiologic and functional relevance of SM deacylase to the ceramide deficiency in the epidermis of patients with AD, we have determined whether the major metabolic intermediate, SPC, that is produced actually accumulates in the stratum corneum of patients with AD as a result of SM deacylase activity. SPC was purchased from Matreya Inc. (Pleasant Gap, PA). Sphingosine (Sph), glucosylsphingosine (GS), and ceramides were obtained from Sigma Chemical Co. (Saint Louis, MO). [Methyl-14C]acetic anhydride was purchased from Amersham. All other chemicals were of reagent grade. Diagnoses of AD were made according to the criteria of Hanifin and Rajka (8Hanifin J.M. Rajka G. Diagnostic features of atopic dermatitis.Acta. Derm. Venereol. (Stockh). 1989; 92: 44-47Google Scholar). Specimens of stratum corneum were obtained from normal forearm skin of healthy individuals (aged from 18 to 31 years old) and from uninvolved and from involved skin of AD patients (aged from 14 to 33 years old) by tape stripping three times with adhesive tape (P.P.S. Nichiban, Tokyo Japan) in the same region. Such tapes were stored frozen at −80°C until use. In this study, erythematous lesions without exudation and swelling were chosen as the involved skin of patients with AD for the preparation of the stratum corneum. The study was approved by the Ethics Committee in Tokyo Women's Medical University. Informed consent was obtained from each patient prior to sampling. After extraction by acetone from human forearm skin, 10 mg of stratum corneum lipids were solubilized in chloroform-methanol (2:1, v/v) and loaded on a 1 ml Silica column (Sep-Pak). After a 4 ml wash with chloroform following a 3 ml wash with acetone, SPC was eluted with 5.6 ml 0.1 N NaOH / 80% methanol and was then extracted by the addition of 4.5 ml chloroform and 2.95 ml water. The lower phase was evaporated under N2 and was then resuspended in small volumes of chloroform-methanol (2:1, v/v) and applied to silica gel 60 HPTLC plates (Merck, KGaA Darmstadt, Germany). The plates were developed in chloroform-methanol-acetic acid-water (50:30:8:5, v/v/v/v) in a horizontal HPLC chamber (CAMAG, Muttenz, Switzerland). Spots on HPTLC plates corresponding to the SPC standard were scraped and dissolved in 100 μl water and approximately 3.4 pg lipids (0.68 ppm × 5 μl) were then subjected to HPLC-mass spectrometry, according to the method of Suzuki et al (9Suzuki M. Sekine M. Yamakawa T. Suzuki A. High-performance liquid chromatography mass spectrometry of glycosphingolipids: I. Structural characterization of molecular species of GlcCer and IV3 bGal-Gb4 Cer.J. Biochem. 1989; 105: 829-833Google Scholar). A JMN-HX110 double focusing mass spectrometer equipped with a fast atom bombardment (FAB) ion source and a JMA-DA5000 data system (JEOL, Tokyo) was used. The accelerating voltage was 6.0 kV and the primary beam for bombardment was 6.0 kV Xe. The ion source temperature was maintained at 40°C. Liquid nitrogen was introduced into the trap of the oil diffusion pump. Mass chromatography was performed to repeat the detection of ions from 300 m/z to 2,200 m/z at intervals of 5 s. HPLC was performed with a Model 120A (Applied Biosystems) on a reversed-phase column of SPHERI-5 (RP-18, 250 mm × 1 mm id, Applied Biosystems) with methanol containing 0.1% trifluoroacetic acid (TFA) and 1% glycerol (G) as the elution solvent at a flow rate of 100 ml per min. The column oven temperature was kept at 40°C. A frit interface with a splitter (JEOL) was set up between the HPLC and the MS. The split ratio was 1:19; therefore, one-twentieth of each injected sample was introduced into the FAB/MS and the rest was discarded via a drainage tube. SPC, GS, or Sph was quantified using the modified method of Yatomi et al (10Yatomi Y. Ruan F. Ohta J. Welch R.J. Hakomori S. Igarashi Y. Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride.Anal. Biochem. 1995; 230: 315-320Google Scholar). Each dried sample (20–50 μg) was dissolved in 20 μl 0.008 N NaOH in dehydrated methanol. The acetylation reaction as depicted in Fig. 1Awas started by the addition of 20 μl 10 mM [methyl-14C]acetic anhydride in chloroform and proceeded for 2 h at 37°C. The remaining anhydride was hydrolyzed by the addition of 200 μl 0.2 N NaOH in methanol and incubation at room temperature. The [14C]C2-glucosylceramide and SM or cer-amide produced, which were identified using each standard, was extracted by adding 0.78 ml methanol, 0.98 ml chloroform, and 0.9 ml 1N KCl. Carrier lipids were applied to the solvent to increase the efficiency of extraction. The resultant lower phase was evaporated under N2 and was then resuspended in a small volume of chloroform-methanol (2:1, v/v) and applied to silica gel 60 HPTLC plates (Merck). The plates were developed in chloroform-methanol-acetic acid (190:9:1, v/v/v) for C-2 ceramide, chloroform-methanol-acetone (76:20:4, v/v/v) for C-2 glucosylceramide, and chloroform-methanol-water (65:25:4, v/v/v) for C-2 sphingomyelin in a horizontal HPLC chamber (CAMAG), and were quantitated using a imaging analyzer (BAS-2000, Fuji Film Corp, Tokyo, Japan). A representative chromatogram and the standard SPC curve are shown in Fig. 1B and Fig. 2. In this experiment using the stratum corneum samples, SPC appears to exist at 5–50 ng per sample. A representative chromatogram and the standard curve for Sph and GS was described elsewhere (11Arikawa J. Ishibashi M. Kawashima M. Takagi Y. Ichikawa Y. Imokawa G. Decreased levels of sphingosine, a natural anti-microbial agent, may be associated with vulnerability of the stratum corneum from patients with atopic dermatitis to colonization by Staphylococcus aureus.J. Invest. Dermatol. 2002; 119: 433-439Google Scholar, 12Ishibashi M. Arikawa J. Okamoto R. Kawashima M. Takagi Y. Oguchi K. Imokawa G. Physiological relevance of another hitherto undiscovered epidermal enzyme, glucosylceramide deacylase to acylceramide deficiency in atopic dermatitis (Abstract).Jpn. J. Dermatol. 2001; 111: 413Google Scholar).Fig. 2The standard curve of SPC.View Large Image Figure ViewerDownload (PPT) To measure ceramide and lysosphingolipids, including SPC, GS and Sph, stratum corneum lipids were extracted with the modified method of Rawlings et al (13Rawlings A.V. Davies A. Carlomusto M. Pillai S. Zhang K. Kosturko R. Verdejo P. Feinberg C. Nguyen L. Chandar P. Effect of lactic acid isomers on keratinocyte ceramide synthesis, stratum corneum lipid levels and stratum corneum barrier function.Arch. Dermatol. Res. 1996; 288: 383-390Google Scholar). In brief, stratum corneum was removed from the volar side of forearm skin by stripping with adhesive tape (P.P.S. Nichiban: 4 × 2.5 cm) three times. Next, the stratum corneum was separated from the adhesive tape by washing with n-hexane under ultrasonication (Bransonic B3200, Yamato Scientific Co.). The separated stratum corneum was dried and weighted (0.5–1.0 mg per sample) to express SCP amount as ng/mg stratum corneum weight. Sphingolipids, including ceramides, SPC, GS, and Sph, were extracted from the separated stratum corneum with chloroform-methanol (2:1, v/v) and were subjected to quantitation of ceramides, SPC, GS, and Sph. For quatitation of ceramides, approximately 30 μg of the extracted lipids was applied to thin-layer chromatograms (TLC), which was developed twice with chloroform-methanol-acetic acid (190:9:1, v/v/v) to resolve ceramides. After solvent development, the chromatograms were air-dried, sprayed with 10% CuSO3, 8% H3PO4 aqueous solution, and were then charred on a 180°C hotplate. The charred lipids were quantitated by photodensitometry (Shimazu CS-9000) and the data were subjected to 2-Dimensional Image Analyzer (Shimazu). Ceramides were quantitated by determining the micrograms of ceramides on a TLC chart from appropriate commercial standards, and are expressed as μg ceramide/mg stratum corneum weight. Ceramides (non-hydroxy fatty acid and hydroxy fatty acid type) (Sigma Chemical Co.) were used as standards for ceramides 1 and 2, and ceramides 3, 4, 5, and 6, respectively. The reproducibility of this method was confirmed using triplicate samples from the same subjects and the deviation of values (total lipids or micrograms ceramide per milligrams stratum corneum) was within 5% of the means. A nonparametric one-way ANOVA (Kruskal-Wallis test) was used to evaluate differences between groups. Where appropriate, a nonparametric post hoc multiple comparison test (Steel-Dwass test) was performed to evaluate differences between the groups. P < 0.05 was considered statistically significant. To determine whether SPC exists in the stratum corneum of healthy subjects at substantial levels, HPLC-MS spectrometry was carried out using lipids extracted from the stratum corneum following their separation by TLC (Fig. 3A). Figure 3Bshows mass chromatograms of purified Sph-PC (20 ng) injected into the LC-MS, and peaks selected as the pseudo-molecular ions ([M-H]−) of the molecular species of Sph-PC at m/z 184 and m/z 465 were observed clearly. As shown in Fig. 3C, the mass spectrum of each of these molecular species was obtained and pseudo-molecular ions were detected with strong intensity at m/z 180 and m/z 456. Furthermore, ions due to the elimination of sphingosine from the molecules were also detected at m/z 184, although the intensity of those ions was rather weak. Mass chromatograms of the TLC-separated lipids (Fig. 3D)revealed that there are two peaks at 184 m/z and 465 m/z that correspond in molecular weight to PC and SPC, respectively. This chromatographic pattern is in agreement with that observed using the SPC standard (Fig. 3B) in which there are two similar peaks at 184 m/z and 465 m/z, which correspond in molecular weight to PC and SPC, respectively.Fig. 3BMass chromatograms of the molecular species of an SPC standard. SPC was dissolved in 100 μl of water-methanol (2:8, v/v) and 0.2 μl of the solution was injected into the LC-MS.View Large Image Figure ViewerDownload (PPT)Fig. 3CMass spectra of the molecular species of SPC standard.View Large Image Figure ViewerDownload (PPT)Fig. 3D:Mass chromatograms of the molecular species of SPC separated by TLC from lipids that had been extracted from healthy forearm skin.View Large Image Figure ViewerDownload (PPT) Quantitative analysis of SPC in the stratum corneum of patients with AD revealed that there is a significant increase (148% for non-lesion and 297% for lesion, n = 40–47, P < 0.01)) in the content of SPC/mg stratum corneum in the uninvolved and in the involved stratum corneum compared with age-matched healthy controls (Fig. 4A). In contrast, there was no increase in the SPC content in the involved stratum corneum of patients with chronic eczema, which suggests that SPC is not upregulated as a simple result of the inflammation seen in AD. In a parallel study in which quantitative analysis of ceramides was also carried out using stratum corneum samples of a similar group of patients with AD, there was a significant decrease in the content of ceramide/mg stratum corneum (n = 53–62, P < 0.01) in both the uninvolved and in the involved stratum corneum compared with age-matched healthy controls (Fig. 4B). Thus, there is a reciprocal relationship between SPC and ceramide levels among healthy controls, uninvolved and involved skin of patients with AD.Fig. 4The quantitation of SPC (A) and ceramides (B) in the stratum corneum from healthy controls and from patients with atopic dermatitis (AD) or chronic eczema. A: SPC measurement: age-matched control, n = 40, average 24.3 years old; uninvolved skin of AD, n = 47, average 22.3 years old; involved skin of AD, n = 44, average 21.7 years old; chronic eczema, n = 6, average 49.8 years old. B: Ceramide measurements: Age-matched control, n = 53, average 25.5 years old; uninvolved skin of AD, n = 62, average 21.4 years old; involved skin of AD, n = 56, average 20.5 years old.View Large Image Figure ViewerDownload (PPT) Comparison between the amounts of ceramide and SPC in the same individuals (Fig. 5A)demonstrated that there is a weak inverse relationship (n = 51, r = −0.44, P = 0.0012) between levels of ceramides and SPC that accumulate in the stratum corneum. Among control, uninvolved, and involved groups, there is the inverse relationship (n = 13, r = −0.641, P = 0.018) only in the involved AD group (Table 1). In this group used for correlation analysis, there is a significant decrease in the amounts of ceramide in the uninvolved and involved stratum corneum from patients with AD compared with healthy control (Table 2). In comparison with another lysosphingolipid, GS, which is derived from glucosylceramide by another novel epidermal enzyme termed glucosylceramide deacylase, which is significantly increased in the stratum corneum of patients with AD (12Ishibashi M. Arikawa J. Okamoto R. Kawashima M. Takagi Y. Oguchi K. Imokawa G. Physiological relevance of another hitherto undiscovered epidermal enzyme, glucosylceramide deacylase to acylceramide deficiency in atopic dermatitis (Abstract).Jpn. J. Dermatol. 2001; 111: 413Google Scholar), there is a significant positive correlation (n = 30, r = 0.703, P < 0.01) between levels of SPC and GS (Fig. 5B). Among control, uninvolved, and involved groups, there is a significant positive correlation (n = 11, r = 0.687, P = 0.020) only in the involved AD group (Table 1). In this group used for correlation analysis there is a significant increase in the amounts of glucosylsphingosine in the uninvolved and involved stratum corneum from patients with AD compared with healthy control (Table 2). In contrast, comparison with Sph, which is a degradative product from ceramide by ceramidase, demonstrated that there is no correlation (n = 32, r = −0.182, P = 0.319.) between levels of SPC and Sph in the overall group (Fig. 5C) as well as the individual groups (Table 1). In this group used for correlation analysis, there is a significant decrease in the amounts of sphingosine in the uninvolved and involved stratum corneum from patients with AD compared with healthy control (Table 2).Fig. 5The relationship between SPC and other sphingolipids in the stratum corneum of some of the patients shown in Fig. 4. A: SPC and ceramide. n = 51, The levels of SPC and ceramide were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 20), or from uninvolved skin (n = 18) or involved skin (n = 13) of patients with AD. B: SPC and GS, n = 30. The levels of SPC and GS were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 8), or from uninvolved skin (n = 11) or involved skin (n = 11) of patients with AD. C: SPC and Sph, n = 32. The levels of SPC and Sph were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 9), or from uninvolved skin (n = 12) or involved skin (n = 11) of patients with AD.View Large Image Figure ViewerDownload (PPT)Fig. 5The relationship between SPC and other sphingolipids in the stratum corneum of some of the patients shown in Fig. 4. A: SPC and ceramide. n = 51, The levels of SPC and ceramide were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 20), or from uninvolved skin (n = 18) or involved skin (n = 13) of patients with AD. B: SPC and GS, n = 30. The levels of SPC and GS were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 8), or from uninvolved skin (n = 11) or involved skin (n = 11) of patients with AD. C: SPC and Sph, n = 32. The levels of SPC and Sph were simultaneously measured as described in the Materials and Methods using the stratum corneum from healthy control skin (n = 9), or from uninvolved skin (n = 12) or involved skin (n = 11) of patients with AD.View Large Image Figure ViewerDownload (PPT)TABLE 1Correlation of SPC with ceramide, glucosylsphingosine and sphingosine in the same groupsCounterpart to SphingosylphosphorylcholineControlAD UninvolvedAD InvolvedOverallCeramiden = 20 n = 18n = 13 n = 51 r = −0.275 r = −0.303 r = −0.641 r = −0.44P = 0.210 P = 0.222P = 0.018 P = 0.0012Glucosylsphingosinen = 8 n = 11n = 11 n = 30 r = 0.397 r = 0.427 r = 0.687 r = 0.703P = 0.330 P = 0.190P = 0.020 P = 0.0000Sphingosinen = 9 n = 12n = 11 n = 32 r = 0.293 r = −0.216 r = −0.050 r = −0.182P = 0.444 P = 0.499P = 0.882 P = 0.319 Open table in a new tab TABLE 2Comparison in the amounts of ceramide, glucosylsphingosine and sphingosine in the same groups used for correlation analysisControlAD UninvolvedAD Involvedμg/mg stratum corneumCeramide27.94 ± 11.9021.03 ± 10.85aP < 0.05.17.44 ± 7.91cP < 0.005.n = 20n = 18n = 13(100%)(75.3%)(63.4%)ng/mg stratum corneumGlucosylsphingosine 8.05 ± 6.86 13.37 ± 8.87aP < 0.05.22.74 ± 19.78aP < 0.05.n = 8n = 11n = 11(100%)(166.1%)(282.5%)Sphingosine1578.91 ± 679.01954.47 ± 381.55bP < 0.01.937.59 ± 678.47aP < 0.05.n = 9n = 12n = 11(100%)(60.5%)(59.4%)a P < 0.05.b P < 0.01.c P < 0.005. Open table in a new tab To characterize the physiological role of SM deacylase in the epidermis of patients with AD, it was important to determine whether its enzymatic reaction product, SPC, is released into the epidermis and accumulates in the stratum corneum. By means of HPLC-mass chromatography, we were able to demonstrate that SPC exists in the superficial stratum corneum of healthy control skin. Since there are no known biological pathways that might lead to the generation of SPC other than SM deacylase (14Desai N.N. Carlson R.O. Mattie M.E. Olivera A. Buckley N.E. Seki T. Brooker G. Spiegel S. Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.J. Cell Biol. 1993; 121: 1385-1395Google Scholar), this suggests that SM deacylase functions to some extent even in the epidermis of healthy controls. This is also substantiated by our previous report (7Hara J. Higuchi K. Okamoto R. Kawashima M. Imokawa G. High-expression of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis.J. Invest. Dermatol. 2000; 115: 406-413Google Scholar), which described a low but significant level of SM deacylase activity in the epidermis of healthy controls. The existence of SPC in the stratum corneum further suggests that SPC generated from SM by the action of SM deacylase (probably in the interface between the granular and the stratum corneum layers) is transferred to and eventually accumulates in the stratum corneum during the keratinization process. The present quantitative analysis of SPC in the stratum corneum of patients with AD demonstrated that there is a significant up-regulation in the amount of SPC in uninvolved and involved stratum corneum from patients with AD compared with age-matched healthy controls. This contrasts with no increase in SPC in the involved stratum corneum of patients with chronic eczema, which suggests that the up-regulation of SPC in AD does not result from ordinary inflammation, but is associated with the altered lipid metabolism characteristic for AD. Parallel analysis of ceramides revealed that while there is a significant decrease in the content of ceramides in uninvolved and in involved stratum corneum of patients with AD, which is consistent with results previously reported (5Imokawa G. Abe A. Jin K. Higaki Y. Kawashima M. Hidano A. Decreased level of ceramides in stratum corneum of atopic dermatitis: An etiologic factor in atopic dry skin?.J. Invest. Dermatol. 1991; 96: 523-526Google Scholar), there is a reciprocal relationship between the increase in SPC and the decrease in ceramide in the stratum corneum of healthy controls, and of lesional and non-lesional skin from patients with AD. This inverse relationship between SPC and ceramides allows us to assume that the ceramide deficiency in AD can mainly be attributed to physiological factors responsible for the increased generation of SPC, which so far is thought to result only from the action of the recently discovered epidermal enzyme, SM deacylase (6Murata Y. Ogata J. Higaki Y. Kawashima M. Yada Y. Higuchi K. Tsuchiya T. Kawaminami S. Imokawa G. Abnormal expression of sphingomyelin acylase in atopic dermatitis: An etiologic factor for ceramide deficiency?.J. Invest. Dermatol. 1996; 106: 1242-1249Google Scholar). As novel properties of SM deacylase that are distinct from other deacylase enzymes reported by Ito et al (15Ito M. Kurita T. Kita K. A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms.J. Biol. Chem. 1995; 270: 24370-24374Google Scholar), we have already characterized several enzymatic characteristics of this enzyme as follows (16Higuchi K. Hara J. Okamoto R. Kawashima M. Imokawa G. The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.Biochem. J. 2000; 350: 747-756Google Scholar); 1) using [palmitic acid-1-14C]SM as a substrate, a sharp pH dependency was found for its catalytic activity, with a peak at pH 5.0; 2) in contrast to the molecular weight of sphingolipid ceramide N-deacylase (52,000) found in bacteria (15Ito M. Kurita T. Kita K. A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms.J. Biol. Chem. 1995; 270: 24370-24374Google Scholar), preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis using AD epidermis extracts revealed that the molecular weight of SM deacylase is 40,000 (16Higuchi K. Hara J. Okamoto R. Kawashima M. Imokawa G. The skin of atopic dermatitis patients contains a novel enzyme, glucosylceramide sphingomyelin deacylase, which cleaves the N-acyl linkage of sphingomyelin and glucosylceramide.Biochem. J. 2000; 350: 747-756Google Scholar); 3) analytical isoelectric focusing (IEF) chromatography demonstrated that the pI values of SM deacylase, β-glucocerebrosidase (GlCdase), SMase, and acid ceramidase (CDase) were 4.2, 7.4, 7.0, and 5.7, respectively, again suggesting that these enzymes and sphingolipid ceramide N-deacylase (with a pI = 6.9–7.4) are different from each other; 4) enzymatic analysis using the pI 4.2 protein partially purified by IEF, which contained no contamination with acid CDase, GlCdase, or SMase, showed that radio-labeled SPC was enzymatically liberated from [choline-methyl-14C]SM used as a substrate; and 5) the pI 4.2 protein purified from the stratum corneum extract of AD patients could hydrolyze N-[palmitoyl-1-14C]SM, but not N-[palmitoyl-1-14C]Cer, thus indicating that the SM deacylase is distinct from any known CDases or N-deacylases. These unique enzymatic properties allow us to assume that there is a new identity of this SM deacylase enzyme in the epidermis of patients with AD and that the expression and subsequent function of this novel enzyme is eventual
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