Unravelling new pathways of sterol metabolism: lessons learned from in-born errors and cancer.

PURPOSE OF REVIEW: To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. RECENT FINDINGS: Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), has recently been shown to be metabolized to the corresponding bile acid, 3ß,5α,6ß-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3ß,5α,6ß-triol is likely to be 3ß-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. SUMMARY: Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

On the formation of 7-ketocholesterol from 7-dehydrocholesterol in patients with CTX and SLO.

A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX.

A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.

We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.

Prenatal presentation and diagnostic evaluation of suspected Smith-Lemli-Opitz (RSH) syndrome.

Smith-Lemli-Opitz (SLOS), or RSH syndrome, is an autosomal recessive deficiency of 7-dehydrocholesterol reductase (DHCR7) resulting in an accumulation of 7- and 8-dehydrocholesterol (7- and 8-DHC) in tissues and body fluids. At birth patients have variable malformations of CNS, heart, kidney, genitalia, and limbs, which may be life-limiting. In later course, psychomotor and mental retardation and behavior abnormalities become evident. Prenatally SLOS can be suspected on the basis of malformations and intrauterine growth retardation (IUGR) in prenatal ultrasonography and reduced maternal free estriol in serum. The diagnosis is confirmed by sterol analysis in a chorionic villus biopsy or amniotic fluid (AF). In this study, we evaluated the predictive value of the above mentioned criteria in combination with family history by quantification of sterols in AF in pregnancies with either a family history, ultrasonographical abnormalities typical for SLOS, or reduced maternal serum unconjugated estriol (MSuE3). The relative frequency of SLOS in fetuses with an affected sibling was 0.23, as to be expected for an autosomal recessive disease. The probability for SLOS was <0.6% when neither an affected sib nor more than one typical SLOS malformation was present. For safety reasons and for cost-effectiveness we recommend careful evaluation of history, MSuE3, and clinical presentation before determining sterols in AF.

Sterol profiles in plasma and erythrocyte membranes in patients with Smith-Lemli-Opitz syndrome: a six-year experience.

BACKGROUND: This study reports our experience over the last six years in the diagnosis of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol biosynthesis. METHODS: Gas chromatography/mass spectrometry was used to obtain sterol profiles in plasma and erythrocyte membranes of suspected patients. RESULTS: Plasma sterol reference values calculated in unaffected subjects (n=276) were in agreement with those previously reported. Among patients investigated from 2005 to 2010, we report 16 patients affected by Smith-Lemli-Opitz syndrome, three of whom represent new cases and 13 of whom were follow-up patients. In this period we also identified a new case of chondrodysplasia punctata 2 X-linked. The estimated incidence obtained for Smith-Lemli-Opitz syndrome was 1:93 suspected patients (1.08%). We also studied the effect of storage on the dehydrocholesterols/cholesterol ratio in plasma and erythrocyte membranes of patients affected by Smith-Lemli-Opitz syndrome stored at -20°C for up to 22 and 20 months, respectively. A significant negative linear correlation between storage time and the dehydrocholesterols/cholesterol ratio was identified in both plasma and erythrocyte membranes. The decrease in the dehydrocholesterols/cholesterol ratio in erythrocyte membranes was at least two-fold higher than in plasma. CONCLUSIONS: The results of this study may be helpful for diagnosis and interpretation of data in patients with findings suggestive of a cholesterol biosynthesis defect.

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Major fetal abnormalities associated with positive screening tests for Smith-Lemli-Opitz syndrome (SLOS).

OBJECTIVE: Determine the relationship between positive screening interpretations for Smith-Lemli-Opitz syndrome (SLOS) and other fetal abnormalities, to aid counseling and diagnostic activities. METHODS: An SLOS screening algorithm was incorporated into California's second-trimester screening program for Down syndrome and open neural tube defects (ONTDs). Between 2002 and 2004, 777 088 pregnant women were given an SLOS risk interpretation, using alpha-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotrophin (hCG) measurements. Outcomes were obtained in 98.8% of screen-positive pregnancies. RESULTS: SLOS screen positives, alone or in combination with screen positives for other fetal disorders (Down syndrome, trisomy 18, ONTD), were associated with a high risk for fetal pathology. Type and frequency of chromosomal or anatomic abnormalities (or fetal death) varied according to screen-positive combination. Among 2018 screen-positive pregnancies, 644 fetal deaths were identified. Among the 1374 viable pregnancies, 519 were screen positive for SLOS alone; two SLOS cases and 51 other serious abnormalities were identified (14 aneuploidies; 37 anatomic). The remaining 855 were also screen positive for at least one other disorder; two SLOS cases and 327 other abnormalities were identified (180 aneuploidies; 157 anatomic). CONCLUSION: For screening programs implementing the SLOS algorithm, the present data may be useful for counseling and to guide diagnostic studies.

Maternal apo E genotype is a modifier of the Smith-Lemli-Opitz syndrome.

BACKGROUND: Smith-Lemli-Opitz syndrome (MIM 270400) is an autosomal recessive malformation and mental retardation syndrome that ranges in clinical severity from minimal dysmorphism and mild mental retardation to severe congenital anomalies and intrauterine death. Smith-Lemli-Opitz syndrome is caused by mutations in the Delta7 sterol-reductase gene (DHCR7; EC 1.3.1.21), which impair endogenous cholesterol biosynthesis and make the growing embryo dependent on exogenous (maternal) sources of cholesterol. We have investigated whether apolipoprotein E, a major component of the cholesterol transport system in human beings, is a modifier of the clinical severity of Smith-Lemli-Opitz syndrome. METHOD: Common apo E, DHCR7, and LDLR genotypes were determined in 137 biochemically characterised patients with Smith-Lemli-Opitz syndrome and 59 of their parents. RESULTS: There was a significant correlation between patients' clinical severity scores and maternal apo E genotypes (p = 0.028) but not between severity scores and patients' or paternal apo E genotypes. In line with their effects on serum cholesterol levels, the maternal apo epsilon2 genotypes were associated with a severe Smith-Lemli-Opitz syndrome phenotype, whereas apo E genotypes without the epsilon2 allele were associated with a milder phenotype. The correlation of maternal apo E genotype with disease severity persisted after stratification for DHCR7 genotype. There was no association of Smith-Lemli-Opitz syndrome severity with LDLR gene variation. CONCLUSIONS: These results suggest that the efficiency of cholesterol transport from the mother to the embryo is affected by the maternal apo E genotype and extend the role of apo E and its disease associations to modulation of embryonic development and malformations.

Diagnosis of Smith-Lemli-Opitz syndrome by ultraviolet spectrophotometry

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder due to an inborn error of cholesterol metabolism, characterized by congenital malformations, dysmorphism of multiple organs, mental retardation and delayed neuropsychomotor development resulting from cholesterol biosynthesis deficiency. A defect in 3ß-hydroxysteroid-delta7-reductase (delta7-sterol-reductase), responsible for the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, causes an increase in 7-DHC and frequently reduces plasma cholesterol levels. The clinical diagnosis of SLOS cannot always be conclusive because of the remarkable variability of clinical expression of the disorder. Thus, confirmation by the measurement of plasma 7-DHC levels is needed. In the present study, we used a simple, fast, and selective method based on ultraviolet spectrophotometry to measure 7-DHC in order to diagnose SLOS. 7-DHC was extracted serially from 200 æl plasma with ethanol and n-hexane and the absorbance at 234 and 282 nm was determined. The method was applied to negative control plasma samples from 23 normal individuals and from 6 cases of suspected SLOS. The method was adequate and reliable and 2 SLOS cases were diagnosed

A rapid screening procedure for cholesterol and dehydrocholesterol by electrospray ionization tandem mass spectrometry.

The mono-(dimethylaminoethyl) succinyl (MDMAES) ester is a new derivative for rapid, mild, and sensitive electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of cholesterol and dehydrocholesterol. It is an order of magnitude more sensitive than the previous most practical alternative, the N-methylpyridyl ether derivative. The MDMAES derivative was used to develop a rapid screening procedure for the biochemical diagnosis of Smith-Lemli-Opitz syndrome (SLOS) by measuring the dehydrocholesterol/cholesterol ratio in plasma (5 microl) and plasma spotted onto filter paper. Details of the synthesis of [25,26,26,26,27,27,27-(2)H7]-7-dehydrocholesterol, used as a standard for quantitation, are included. The measurement of total sterols as MDMAES esters, after base hydrolysis of plasma, afforded a dehydrocholesterol/cholesterol ratio of 0.05-2.95 for SLOS patient samples (n = 5) compared with 0.001-0.003 for normal adult controls (n = 20). Direct hexane extraction of plasma without base hydrolysis enabled the measurement of free sterols with a total sample analysis time of <1 h. The free dehydrocholesterol/cholesterol ratio was 0.10-4.47 for SLOS patient samples (n = 5) and 0.003-0.011 for normal adult controls (n = 20).

Smith-Lemli-Opitz syndrome and other sterol disorders among Finns with developmental disabilities.

Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol metabolism in which 7- and 8-dehydrocholesterols are accumulated in blood and tissues. Diagnosis of SLOS and other disorders in cholesterol metabolism (eg, cerebrotendinous xanthomatosis, phytosterolemia, desmosterolosis, and X-linked dominant Conradi-Hünermann-Happle syndrome) can be performed by gas-liquid chromatographic analysis of serum sterols. To elucidate their involvement in developmental disability, we evaluated serum sterols in two study groups: developmentally disabled subjects in long-term care (N = 322) and newborns and young children (N = 49) with features of SLOS in the Finnish population of 5 million. Only 1 SLOS case (type II) was found from among the 49 children. Seven additional adult cases (type I) with a wide range of clinical features and the serum sterol abnormalities characteristic of SLOS were detected from among the developmentally disabled subjects. The frequency of SLOS in the latter group was relatively high (7 in 322). No other hereditary sterol disorders were found, but two subgroups with low serum cholesterol precursor sterols and high serum plant sterols were identified. Several subjects, including the 7 SLOS patients, used ample medication and had abnormalities in serum sterol concentrations. Thus, among the subjects taking melperone, a high serum delta8-cholestenol level suggests an interference by the drug with cholesterol synthesis. Our results emphasize the importance of analyzing the serum sterols of developmentally disabled subjects to diagnose SLOS and of finding putative undiagnosed disorders in sterol metabolism associated with these clinical conditions.

Characterization of photosensitivity in the Smith-Lemli-Opitz syndrome: a new congenital photosensitivity syndrome.

Photosensitivity has recently been reported as a feature of the Smith-Lemli-Opitz syndrome (SLO). The aim of this study was to establish the photobiological features of this disorder and to examine the hypothesis that the photosensitivity is caused by the high levels of 7-dehydrocholesterol found in SLO. All known cases of SLO in the U.K. were reviewed and clinical details of photosensitivity were recorded in detail. The action spectrum of the photosensitive eruption was defined by monochromator light testing. Thirteen of the 23 subjects (57%) had severe photosensitivity, and in 10 there was no photosensitivity. No correlation was identified between levels of 7-dehydrocholesterol and severity of photosensitivity, suggesting that the photosensitivity in SLO is not caused by a direct phototoxic effect mediated by 7-dehydrocholesterol. A novel pattern of photosensitivity was observed, with onset of a sunburn-like erythema on sun-exposed skin within minutes of sun exposure, which persisted in most cases for up to 24-48 h before fading. Monochromator light testing in three subjects showed an ultraviolet (UV) A-mediated photosensitivity eruption with greatest photosensitivity at 350 nm. Photosensitivity is a common and prominent feature of SLO and appears to be UVA-mediated. Elucidation of its biochemical basis may provide insight into normal cutaneous protective mechanisms against UVA-induced photodamage, and also sun sensitivity in general.

Direct analysis of filter paper blood specimens for identification of Smith-Lemli-Opitz syndrome using time-of-flight secondary ion mass spectrometry.

In this study, time-of-flight secondary ion mass spectrometry was used to distinguish between blood of normal infants and that of individuals with Smith-Lemli-Opitz (SLO) syndrome. SLO syndrome results in an abnormally low concentration of blood cholesterol and an elevated concentration of 7-dehydrocholesterol. Blood was spotted on filter paper and analyzed directly with no extractions or separations. Results showed that using ratios of fragment ions for cholesterol/dehydrocholesterol, patients with SLO and normal individuals could be unambiguously distinguished. Unknown samples from 28 individuals were obtained and identified correctly.