Simplified method of determination of serum cholesterol sulfate by reverse phase thin-layer chromatography

A new method for determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) from 1 ml serum by reverse phase thin-layer chromatography (TLC) is described. The method comprises an isolation step of sulfated steroids by means of octadecylsilane-bonded (C18) reverse phase column chromatography, a solvolysis step for desulfation of sulfated steroids, and a C18 TLC step for measurement on a photodensitometer. This method is much simpler and more rapid than the methods previously reported, since neither a radioisotope is needed, nor any steps of saponification, derivatization, tedious scraping from a TLC plate, and time-consuming conventional column chromatography are not required. The present method allowed us to distinguish recessive X-linked ichthyosis (RXLI) very easily from ichthyosis vulgaris (IV) by the size and gradation of clearly visible blue chromogen derived from CS on a TLC plate in RXLI. By photodensitometer scanning, the CS levels in patients with RXLI were about 10 times higher than those of patients with IV and healthy subjects, whereas the DHEAS level was normal in the RXLI patients. The present simplified method proved to be useful in diagnosis of RXLI.     

鱼鳞病患者鳞屑、皮肤和血液的胆固醇和胆固醇硫酸酯分析

目的：探讨脂质与鳞屑性疾病的关系。方法：用气相质谱方法对性联隐性鱼鳞病（ＲＸＬＩ）、寻常性鱼鳞病（ＩＶ）各５例和５例正常对照组的鳞屑、表皮、真皮和血液中的胆固醇硫酸酯（ＣＳ）和胆固醇（ＣＨ）进行了定量分析。结果：鳞屑中ＲＸＬＩ组ＣＳ含量显著高于ＩＶ和正常对照组，ＣＨ和ＣＨ／ＣＳ显著低于其他二组。在表皮、真皮和血液中，ＲＸＬＩ组ＣＳ含量显著增高，而ＣＨ含量无明显变化，ＣＨ／ＣＳ下降。结论：ＲＸＬＩ的鳞屑产生与ＣＳ积聚及ＣＨ／ＣＳ下降有关，而ＩＶ的发病则与ＣＳ和ＣＨ无关。     

X-linked ichthyosis: relation between cholesterol sulphate, dehydroepiandrosterone sulphate and patient's age

Steroid sulphatase deficiency is a feature of recessive X-linked ichthyosis (RXLI) that causes the accumulation of sulphated steroids (SS) in various organs and cells. In a previous study, we detected elevated cholesterol sulphate (CS) and dehydroepiandrosterone sulphate (DHEAS) serum levels in a group of 15 RXLI patients selected in a narrow age range. In the present study both CS and DHEAS serum levels were qualitatively and quantitatively determined using gas-chromatographic analysis in a group of 33 RXLI patients ranging in age from 3 to 70 years. The levels of CS and DHEAS were significantly increased in all patients. Variations in SS were related both to patients' ages and clinical course of the disease. Serum SS levels start to increase in early infancy, peak at puberty, remain elevated in adults and decrease slightly in the elderly.     

Stratum corneum lipid abnormalities in ichthyosis. Detection by a new lipid microanalytical method

Although the biochemical diagnosis of the ichthyoses is still in its infancy, the two recessively inherited types, recessive X-linked ichthyosis (RXLI) and nonbullous congenital ichthyosiform erythroderma (CIE), are accompanied by stratum corneum lipid abnormalities. However, in RXLI, cholesterol sulfate accumulates; in CIE, massive quantities of n-alkanes accumulate. The diagnosis of these disorders has required large quantities of scale for sequential, quantitative thin-layer chromatography (TLC). In this study, we sought to confirm the previously described lipid abnormalities with the use of a rapid, recently developed microchromatographic technique that employs silica gel-coated quartz rods and flame ionization detection (Iatroscan). The cholesterol sulfate content of RXLI (n = 5) scale and the n-alkane content of CIE (n = 8) scale were determined by both TLC and the microchromatographic technique. Less than 10 mg of scale and even single punch biopsy specimens sufficed for the microchromatographic technique, whereas more than 50 mg of scale were required for TLC. Since the microchromatographic technique can rapidly detect diagnostic biochemical abnormalities from readily obtainable, small tissue samples, this method could eventually supplant or supplement standard lipid biochemical techniques for the diagnosis of cutaneous lipidoses.     

Cholesterol sulphate levels in the hair and nails of patients with recessive X-linked ichthyosis

Cholesterol sulphate (CS) has been suggested as an intercellular glue for corneocyte-corneocyte cohesion from studies on patients with recessive X-linked ichthyosis (RXLI). Pathological stratum corneum of RXLI patients was found to show a significant elevation of CS. In the present study hair and nails, unaffected keratinized tissues in RXLI patients, were examined for CS levels. The results demonstrated significantly elevated CS levels in both tissues in RXLI patients (P less than 0.001). In particular the mean CS level in the hair of RXLI patients was five times greater than normal. The present study suggests that hair is a useful material for the diagnosis of RXLI.     

Substrate specific sulfatase activity from hair follicles in recessive X-linked ichthyosis

Recessive X-linked ichthyosis (RXLI) has its biochemical basis in a defect of the enzyme steroid sulfatase. Since several studies have reported a simultaneous deficiency of arylsulfatase C and steroid sulfatase it has been hypothesized that both enzymes are identical. In human hair follicles, however, hydrolytic activity for 4-methylumbelliferone sulfate, the substrate for arylsulfatase C, is found, while dehydroepiandrosterone sulfate is not hydrolyzed at all. These findings suggested the possible existence of two different enzymes. In the present paper structure-activity studies and molecular energy calculations are used for the demonstration that the remaining sulfatase activity in hair follicles of RXLI patients can be explained on the basis of the assumption that the enzyme has not lost its total function but has become less efficient.     

Interactions of cholesterol and cholesterol sulfate with free fatty acids: Possible relevance for the pathogenesis of recessive X-linked ichthyosis

Summary Whereas the stratum corneum contains large amounts of unesterified cholesterol and minimal amounts of cholesterol sulfate, in recessive X-linked ichthyosis (RXLI), levels of cholesterol decrease while cholesterol sulfate content increases. To study the molecular basis for abnormal shedding in RXLI, we compared the interaction of cholesterol and cholesterol sulfate with the free fatty acid, hexadecanoic acid, by differential scanning calorimetry (DSC). While cholesterol and the free fatty acid formed a eutectic mixture, such an interaction did not occur upon mixing of cholesterol sulfate with hexadecanoic acid. In addition, and unexpectedly, free cholesterol appeared to undergo progressive autoxidation during repeated DSC measurements at only slightly supraphysiologic temperatures. These studies may provide a molecular mechanism for the abnormal desquamation that occurs in RXLI. The regular formation of oxidation products of cholesterol observed here, if matched by equivalent molecular events in vivo, may have important implications for epidermal pathophysiology.     

Steroid sulphatase deficiency in patients initially diagnosed as ichthyosis vulgaris or recessive X-linked ichthyosis

Twenty-one patients with ichthyosis were classified as either ichthyosis vulgaris (IV) (five cases) or recessive X-linked ichthyosis (RXLI) (sixteen cases) by using a steroid sulphatase assay of plantar callus and peripheral leukocytes. The patients had presented with various clinical manifestations, which had resulted in some initial misdiagnoses. Cases which initially resemble IV may in fact be RXLI, although we found that if a case is initially diagnosed as RXLI it is unlikely to be a case of IV.     

8-Methoxypsoralen-gas chromatographic determination and serum kinetics

Summary The isolation and gas chromatographic quantitation of 8-methoxypsoralen (8-MOP) is described. The method involves extraction of the drug from serum, thin layer chromatography of the extract and gas liquid chromatography of the eluate from the thin layer plate.Serum concentrations of 8-methoxypsoralen after oral administration have been determined. The drug concentrations were determined by gas chromatography or after administration of tritiated drug, by measuring the radioactivity in serum. Using the gas chromatographic method, maximal serum levels of 0.4–1.5 g 8-MOP per ml were found 70–90 min after oral administration of 50 mg of the drug. Measuring tritium levels in serum, maximal concentration of 0.9 g 8-MOP per ml was calculated, reached 40 min after administration of 20 mg of tritium labelled 8-MOP. Measuring radioactivity, the sensitivity is very high, but the method does not distinguish between the original drug and its metabolic products. The gas chromatographic method, on the other hand, detects the original drug and the results are not affected by the presence of breakdown products.Rapid absorption of the drug from the gastro-intestinal tract was observed following oral administration. The drug is rapidly metabolised and excreted from the body, showing an apparent half life time of about 1.5 h. No detectable drug could be found in serum 10 h after administration when the GLC method was used; however, excretion of radioactive metabolites continued over several days and may indicate a possible accumulation of metabolites, especially in cases when a frequent 8-MOP treatment schedule is applied.Parts of this work were supported by grant No. Scha 213 from the Deutsche Forschungsgemeinschaft, and by the Stiftung Volkswagenwerk (Hannover, Federal Republic of Germany)     

Basis for abnormal desquamation and permeability barrier dysfunction in RXLI

Mutations in the gene for steroid sulfatase (SSase), are responsible for recessive x-linked ichthyosis (RXLI). As a consequence of SSase deficiency, its substrate, cholesterol sulfate (CSO4), accumulates in the epidermis. Accumulation of this amphipathic lipid in the outer epidermis provokes both a typical scaling phenotype and permeability barrier dysfunction. Research on RXLI has illuminated several, potentially overlapping pathogenic mechanisms and provided insights about the role of SSase and CSO4 in normal differentiation, barrier maintenance, and desquamation. We now show here that SSase is concentrated in lamellar bodies (LB), and secreted into the SC interstices, along with other LB-derived lipid hydrolases. There, it degrades CSO4, generating some cholesterol for the barrier, while the progressive decline in CSO4 (a serine protease (SP) inhibitor) permits corneodesmosome (CD) degradation leading to normal desquamation. Two molecular pathways contribute to disease pathogenesis in RXLI: 1) excess CSO4 produces nonlamellar phase separation in the stratum corneum (SC) interstices, explaining the barrier abnormality. 2) The increased CSO4 in the SC interstices inhibit activity sufficiently to delay CD degradation, leading to corneocyte retention. We also show here that increased Ca++ in the SC interstices in RXLI could contribute to corneocyte retention, by increasing CD and interlamellar cohesion. RXLI represents one of the best understood diseases in dermatology--from the gene to the SC interstices, its etiology and pathogenesis are becoming clear, and assessment of disease mechanisms in RXLI led to new insights about the role of SSase and CSO4 in epidermis terminal differentiation.     

Lipoprotein electrophoresis in recessive X-linked ichthyosis

Recessive X-linked ichthyosis (RXLI) is consistently associated with steroid sulphatase deficiency, and a definite diagnosis can be made by measurement of the activity of this enzyme, e.g. in cultured skin fibroblasts and leucocytes. Demonstrating an increased electrophoretic mobility of plasma low-density lipoprotein in RXLI patients has been proposed as a simpler method for the diagnosis of this condition. Our findings in 7 RXLI patients and 7 normal controls confirmed that a discrimination between patients and controls can be obtained by routine lipoprotein electrophoresis. However, due to variation in the results of repetitive performances further studies are needed to evaluate the overall reliability of this diagnostic approach.     

银屑病和鱼鳞病患者血清必需脂肪酸分析

为了探讨必需脂肪酸与鳞屑性皮肤病的关系，用气相质谱法分析了寻常型银屑病，性联隐性鱼鳞病，寻常型鱼鳞病和正常对照组的血清和血清磷脂脂肪酸的组成，结果：银屑病组Ｃ＾４２０、Ｃ＾４２２、Ｃ＾５２２显著低于正常，而Ｃ＾２１８、Ｃ＾３１８及Ｃ＾３２０明显升高。     

Analysis of the STS gene in 40 patients with recessive X‐linked ichthyosis: a high frequency of partial deletions in a Spanish population

Background Recessive X‐linked ichthyosis (RXLI) (OMIM 308100) is a genodermatosis characterized by polygonal, dark, adherent and mild‐to‐moderate scales that normally improve during summer. RXLI is caused by a deficiency in steroid sulphatase (STS), whose gene has been located on the X chromosome (locus Xp22.3). Up to 90% of the mutations described in this gene are complete deletions. Objectives Previous reports of partial deletion of STS gene in cases of RXLI prompted us to determine the incidence of these abnormalities in a Spanish population. Methods We have studied exons 1, 5 and 10 of the STS gene by polymerase chain reaction in 40 patients with clinical features of RXLI. Results Our results revealed that 30 patients presented complete deletions (75%) while 10 patients had partial deletions (25%) a rate higher than that reported in the previous studies. Conclusions Amplification of exons 1, 5 and 10 is reliable in screening RXLI in the population studied here. No correlation was found between phenotype and the extent of the deletions.     

Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins

Two monoclonal antibodies (AKH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts.     

Recessive X‐linked ichthyosis associated with hypertrophic pyloric stenosis: a chance occurrence?

The association of recessive X-linked ichthyosis (RXLI) and hypertrophic pyloric stenosis (HPS) has been considered to be due to a probable contiguous gene defect. However, there are several reports of patients with large deletions on both sides of the steroid sulphatase gene (responsible for RXL1) that show no signs of HPS. We report the third pedigree wherein RXL1 was associated with HPS. Apart from the proband, both diseases showed themselves as independent events in the family tree with ichthyosis present in two other individuals and HPS in three other relatives. We calculated the probability that both diseases occurred simultaneously in the index case as a chance occurrence as 1 : 40 (using the Independence principle of probability). We conclude that in our pedigree it is likely that these two rare diseases show an accidental and not a true genetic association.     

Palmoplantar keratoderma with an unusual composition of stratum corneum and serum sterol derivatives: a new entity?

Summary Little is known about the aetiology and pathogenesis of the different types of inherited and acquired palmoplantar keratodermas. We describe a condition of painful palmoplantar keratoderma with an altered stratum corneum lipid pattern which may be responsible for the excessive cornilication. Plantar stratum corneum lipids were analysed by quantitative thin-layer chromatography. Serum lipids, and the activities and gene loci of the enzymes serum steroid sulphatase and arylsulphatase C were also determined. Examination revealed that both the stratum corneum and the serum cholesterol sulphate (CS) content were significantly elevated in comparison with the stratum corneum cholesterol ester content. The cholesterol content was unchanged compared with controls. Serum activities of steroid sulphatase and arylsulphatase C were decreased, but not to the extent found in recessive X-linked ichthyosis. Their gene loci did not show any deletions.
This unique distribution of stratum corneum sterol derivatives, reflected by the elevated serum CS concentration, may contribute to the altered structural and functional properlies of intercellular lipid lamellae within the stratum corneum of this type of keratoderma.     

Purification of immunoprecipitated pemphigus foliaceus antigen fragment and its use in radioimmunoassay.

A major difficulty in biochemical studies of the pemphigus foliaceus (PF) antigen is the lack of a method for its quantitative determination. Immunofluorescence blocking and immunoprecipitation methods are semiquantitative and time consuming. Radioimmunoassay (RIA) methods are quantitative but they require pure and stable antigen preparations that have not been available for PF. The present investigation shows the further purification of a previously described preparation of PF antigen fragment obtained from trypsinization media of mouse skin (Con A Frn) and demonstrates its usefulness in a RIA method for quantitation of the antigen. The major contaminants of the 45-kD tryptic fragment of the PF antigen (tf-PF) in immunoprecipitates of the Con A Frn with PF sera were identified as H and L chains of murine IgG and mannose-binding lectins. The IgG contaminants could be removed by avoidance of blood contamination during preparation of the Con A Frn and/or pre-absorption of the Con A Frn with protein A Sepharose. The lectins could be removed by affinity chromatography of the Con A Frn on asialofetuin column and washing the immunoprecipitates with 0.2 M alpha-methyl-mannoside. Using the purified, labeled Con A Frn in RIA, we showed that standard curves could be established and the amounts of PF antigen could be determined in different extracts without the need for electrophoresis, autoradiography, or scanning. This RIA method is rapid and can be easily used to analyze many samples, e.g., chromatographic fractions and extracts made from different tissues.     

Microanalytical screening of all major stratum corneum lipids by sequential high-performance thin-layer chromatography

For rapid and sensitive screening of lipid biochemical abnormalities of scaling skin disorders a sequential, one-dimensional high-performance thin-layer chromatographic method (HPTLC) has been developed. All major human stratum corneum lipid classes, i.e., cholesterol sulfate, glucosylceramides, six major ceramide fractions, free sterols, free fatty acids, triglycerides, sterol esters, squalene, and n-alkanes, are separated and quantitated after a stepwise development of a single silica gel 60 HPTLC-plate using three consecutive solvent systems. Reproducible results have been obtained by degradative charring as well as fluorescence detection. By fluorescence detection the method is particularly suitable for the determination of minor amounts of cholesterol sulfate and other sterols.     

Nonsyndromic types of ichthyoses – an update

Ichthyoses are genetically determined Mendelian disorders of cornification (MEDOC) that are characterized by universal scaling. Today we distinguish between non‐syndromic and syndromic forms. Ichthyosis vulgaris is the most frequent type (prevalence 1:100) and is caused by autosomal semi‐dominant filaggrin mutations. It is associated with a higher risk for the development of atopic diseases, such as atopic eczema and allergic rhinitis. Recessive X‐linked ichthyosis (RXLI) occurs almost exclusively in boys; in Germany it has a prevalence of around 1:4,000. It is caused by steroid sulfatase deficiency and is often associated with further clinical problems, such as cryptorchidism (～20%) or social communication deficits, such as attention deficit hyperactivity syndrome (40%) or autism (25%). Autosomal recessive congenital ichthyosis (ARCI) is genetically very heterogeneous and 8 different genes have been identified so far. The most frequent cause of ARCI is a transglutaminase 1 deficiency (prevalence 1:200, 000). Mutations in keratin genes are the cause of the keratinopathic ichthyoses, such as epidermolytic ichthyosis. They manifest at birth and often feature episodes of blistering. Most of these types are inherited as autosomal dominant traits, but autosomal recessive forms have also been described on occasion.     

Cholesterol sulfotransferase of newborn mouse epidermis

Recent studies of the epidermis in patients with recessive X-linked ichthyosis indicate that cholesterol sulfate is an important endogenous substrate for steroid sulfatase in the stratum corneum. We report here that cholesterol sulfotransferase, which converts cholesterol to cholesterol sulfate, is present in the lower living epidermis. Epidermal cytosol also sulfates phenols and some steroids, and such reactions may be important in defending against compounds absorbed percutaneously and in modulating the pharmacologic activity of topical medicaments. Sodium salicylate and sodium citrate inhibit the sulfotransferase activity noncompetitively, and inhibition of cholesterol sulfate formation may be important in the desquamative action of these topical "keratolytics."