Simplified determination of serum cholesterol sulfate by gas-liquid chromatography combined with cyclohexylsilane-bonded phase column purification

Summary A new gas-liquid chromatographic (GLC) determination of cholesterol sulfate (CS) and dehydroepiandrosterone sulfate (DHEAS) for a biochemical diagnosis of recessive X-linked ichthyosis (RXLI) is described. Although the GLC method for determination of CS is known to be more sensitive than the thin layer chromatographic (TLC) method, the former method has not been widely employed because of its complicated pre-purification steps. The present method allows us to measure the serum levels of CS and DHEAS without tedious purification steps such as multiple conventional column chromatography and preparative thin layer chromatography. Sulfated steroids are rapidly purified with a commercially available mini disposable cyclohexylsilane-bonded phase (CH) column, CH BOND ELUT, and the purified steroids after desulfation are converted to water-resistant tert-butyldimethylsilyl ether derivatives for the GLC analysis on dual 2 m glass columns packed with 2% XE-60 on Chromosorb W.By the present method, serum CS concentrations in RXLI patients were shown to be about 10 times higher than those in patients with ichthyosis vulgaris, carriers of RXLI, and healthy subjects. This method is more suitable not only for a biochemical diagnosis of RXLI but also for studies on the metabolism of sulfated steroids than the previous time-consuming GLC methods.     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

Excretion of (sulfated) steroids in the urine and excretion of cholesterol sulfate in the feces of boys with recessive X-linked ichthyosis

Summary The excretion of sulfated steroids was investigated in the urine and feces of six boys aged 9 months to 7 years and 10 months who had recessive X-linked ichthyosis. Profiles of urinary total steroids as well as sulfated steroids were normal. Cholesterol sulfate excretion in the urine was not elevated. In the feces 2–20% of total cholesterol was cholesterol sulfate, whereas in the feces of 28 healthy children no cholesterol sulfate was demonstrable. In the 6 patients total cholesterol excretion (500–2,500 mol/kg feces) was also elevated in comparison with the 28 healthy controls (150–700 mol/kg feces, mean 365 mol/kg feces)     

Metabolic alterations of DHEA and cholesterol sulphates in the hair of patients with acne measured by liquid chromatography–mass spectrometry

Please cite this paper as: Metabolic alterations of DHEA and cholesterol sulphates in the hair of patients with acne measured by liquid chromatography–mass spectrometry. Experimental Dermatology 2010; 19 : 694–696. Abstract: As the hormonal levels in scalp hair reflects the condition of skin appendage, the level of dehydroepiandrosterone‐3‐sulphate (DHEAS) and cholesterol sulphate (CS) was evaluated in scalp hair obtained from patients with acne. The hair samples were extracted by alkaline solution and were analysed by liquid chromatography–mass spectrometry coupled to column switching system. The levels of DHEAS in scalp hair of women with acne were higher (P < 0.001) than controls, while the levels of CS in scalp hair of women and men with acne were higher (P < 0.001) than both control subjects. Increased hair levels of both DHEAS and CS could indicate acne development because of its direct action or stimulatory effect on local enzyme activity. It may be also helpful to understand the pathogenesis of acne based on follicular retention hyperkeratosis and increased sebum production of both steroid sulphates.     

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.     

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。     

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.     

Structural characterization of the skin glycosaminoglycans in patients with pseudoxanthoma elasticum

Background Complex polysaccharides, glycosaminoglycans (GAGs), their amount, and fine structure were determined in the skin (epidermis + dermis) of pseudoxanthoma elasticum (PXE)‐affected patients in comparison with healthy subjects. Methods Nonlesional skin GAGs were extracted and specifically determined by enzymatic treatment and high‐performance liquid chromatography separation. Results Dermatan sulfate (DS) and hyaluronic acid (HA) were found to be the major GAG species in normal subjects, with contents of approximately 20% for DS and 58% for HA. The chondroitin sulfate (CS) content (unsaturated six‐sulfated disaccharide) was approximately 21%. Skin from patients with PXE showed similar HA (61%), DS (22%), and CS (16.7%) contents. No change in the total charge density or nonsulfated/sulfated GAG ratio was noted in PXE‐affected subjects, and no modification of the position of the sulfate groups (4s/6s) on the CS/DS backbone. A significant increase (approximately 88%; P < 0.01) in the total amount of GAGs (HA + DS + CS) was found in the PXE group vs. normal subjects, however. Conclusions In the skin of PXE‐affected patients, the altered metabolic processes produce an increase in the total amount of GAGs able to accumulate salts, in particular calcium ions, within the elastic fibers, producing ion precipitates that affect the organization of the matrix fiber.     

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.     

Treatment of androgenic disorders with dexamethasone: dose-response relationship for suppression of dehydroepiandrosterone sulfate

Glucocorticoids are effective in suppressing androgens in many women whose levels of these steroids are elevated. Their use has been controversial because of inconsistent reports about efficacy and concern about safety. We investigated the dose-response relationship for suppression of dehydroepiandrosterone sulfate (DHEAS) with the use of dexamethasone. Thirty women with an initial DHEAS value of greater than or equal to 300 micrograms/dl were studied. All had cystic or inflammatory acne, hirsutism, or androgenic alopecia. Dexamethasone was given as a single bedtime dosage of 0.125, 0.250, or 0.375 mg. Mean dosage required for suppression was 0.256 mg daily. Suppression of the DHEAS level to less than or equal to 200 micrograms/dl was achieved with 0.125 mg in 25% of women, 0.250 mg in an additional 50%, and 0.375 mg in a further 20%. Most patients were taking spironolactone when the study was performed. Effective suppression is attained with dexamethasone doses significantly lower than previously thought. Use of these doses was not associated with a significant incidence of adverse effects.     

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."     

Mitotic Langerhans cell as a possible sign of activation in ichthyosis

The fine structure of Lagerhans cells (LC) in four rare types of ichthyosis, namely recessive ichthyosis congenita type II, III and IV and ichthyosis hystrix Curth-Macklin was examined. Signs of LC activation were observed in eight of 21 patients. In IC type IV, the rare occurrence of a mitotic LC was observed. It is possible that LCs are secondarily activated in keratinization disorders.     

X-linked ichthyosis and ichthyosis vulgaris: comparison of their clinical features based on biochemical analysis

Thirty cases of X-linked ichthyosis (XLI) and 32 cases of ichthyosis vulgaris (IV) were diagnosed by measuring the steroid sulphatase activity of peripheral blood lymphocytes or the electrophoretic mobility of serum LDL or both. The clinical features of the two conditions were then compared. In both diseases 60-66% of patients had a family history of the condition. Ichthyosis was noted at birth or immediately afterwards in 59% of the patients with XLI while it appeared in infancy in 68% of those with IV. Scales were mostly large and brown or dark brown in patients with XLI, while the majority of patients with IV had small brown or light brown scales. The distribution of the ichthyotic lesions differed in the two types of ichthyosis. On the trunk, the abdomen was more severely involved than the back in 63% of the cases with the XLI, whereas the back was more scaly than the abdomen in 44% of those with IV. On the extremities, the extensor surface was more severely affected than the flexor surface in both types. X-linked ichthyosis was characterized by the presence of lesions in the pre-auricular area, which were found in 93% of the cases with XLI, while only 17% of the IV patients had ichthyotic lesions at this site. Involvement of the preauricular area could be an important clinical feature for distinguishing XLI from IV.