Sun-induced production of vitamin D3 throughout 1 year in tropical and subtropical regions: relationship with latitude, cloudiness, UV-B exposure and solar zenith angle.

This study evaluated the differences in vitamin D3 synthesis in two different latitudes throughout 1 year using an in vitro model, which simulates cutaneous vitamin D photoproduction. Borosilicate ampoules containing 7-dehydrocholesterol (7-DHC) were exposed to sunlight hourly throughout the daylight hours, 1 day per month for a year, in Fortaleza (latitude 03° 43' 01" S-LAT3° S) and Sao Paulo (latitude 23° 32' 53" S-LAT23° S). Later, vitamin D3 and photoisomers of 7-DHC (tachysterol and lumisterol) were measured by a high-performance liquid chromatography system (HPLC). Vitamin D synthesis weighted UV radiation (UVBVitD) and solar zenith angle (SZA) were calculated during the same periods for both latitudes. Vitamin D3 synthesis occurred throughout the year in both locations, as expected in latitudes lower than 35°. Median of photoconversion to vitamin D3 through the year was higher in LAT3°S [median (IQR): LAT 3°S 4.1% (6.0); LAT 23°S 2.9% (4.5); p value = 0.020]. Vitamin D3 production strongly correlated with UV-B (LAT3° S, r = 0.917; p < 0.0001 and at LAT23° S, r = 0.879; p < 0.0001) and SZA (LAT3° S, r = - 0.924; p < 0.0001 and in LAT23°S, r = - 0.808; p < 0.0001). Vitamin D3 production starts later in LAT23° S, especially in winter. Lowest percentages were observed in June in both cities, although, compared to LAT3° S, in LAT 23° S the conversion was over 50% lower in the winter period. Cloudiness impaired photoproduction of Vitamin D3 even in summer months in both latitudes. Our results provide data to help guide medical recommendations for sensible sun exposure to promote the cutaneous production of vitamin D3 at different latitudes, seasonality, time of day and cloudiness status in Brazil.

Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line.

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.

Feeding impairments associated with plasma sterols in Smith-Lemli-Opitz syndrome.

OBJECTIVE: To quantitatively evaluate feeding impairment in children with Smith-Lemli-Opitz syndrome (SLOS) and to correlate feeding impairment with clinical and biochemical indices of disease severity. STUDY DESIGN: The study subjects were 26 children with SLOS ranging in age from 0.4 to 19 years. Clinical severity was measured using an existing scoring system. We created a tool to quantitatively evaluate feeding. Plasma sterol concentrations were measured, and statistical associations (correlations) with feeding scores were calculated. RESULTS: Oral hyposensitivity or hypersensitivity, adverse behaviors, and risk for dysphagia were seen in ∼65% of the children with SLOS. Thirteen of the 26 children experienced failure to thrive, and 10 children required gastrostomy. Plasma concentration of 7-dehydrocholesterol, as a measure of severity, was correlated with total feeding score and oral function subcategory score (P < .001) and less so with oral structure score, adverse behaviors, or dysphagia. Correlations with cholesterol concentrations were less statistically significant. A plasma 7-dehydrocholesterol concentration >0.24 mmol/L or cholesterol concentration <1.95 mmol/L was predictive of gastrostomy tube use. Feeding impairments may improve with age. CONCLUSION: Feeding impairment is common and complex in patients with SLOS. Our findings confirm that oral sensitivities, adverse feeding behaviors, and risk of oral phase dysphagia are amenable to quantitative evaluation and analysis. Feeding difficulties in children with SLOS are correlated with plasma sterol concentrations, suggesting a link between the biochemical severity of SLOS and feeding function. These findings expand the behavioral phenotype of SLOS and begin to provide insight into the biological causes of feeding difficulties.

Development of molecularly imprinted poly(methacrylic acid)/silica for clean-up and selective extraction of cholesterol in milk prior to analysis by HPLC-UV.

In the present paper the assessment of a novel molecularly imprinted polymer, poly(methacrylic acid)/silica, for clean-up and selective extraction of cholesterol in milk samples is described. The relative selectivity coefficient (k) values for cholesterol/5-α-cholestane and cholesterol/7-dehydrocholesterol systems were found to be 5.08 and 6.08, respectively, thus attesting the selectivity of the MIP for cholesterol under competitive adsorption with structurally analogous steroid compounds. The milk analysis was initially based on saponification followed by liquid-liquid extraction with n-hexane. Then, the protocol of molecularly imprinted solid phase extraction (MISPE) was carried out by loading the milk hexanic extract through 200 mg of MIP or NIP (non-imprinted polymer) packed into SPE cartridges at a flow rate of 0.6 mL min(-1). The washing step was performed by using n-hexane followed by further elution with ethanol and HPLC-UV analysis at 208 nm. From the breakthrough curve the maximum adsorption capacity of the MIP towards cholesterol was found to be 29.51 mg g(-1). The precision of the MISPE protocol was assessed as intra- and inter-days yielding RSD (relative standard deviations) lower than 4.10%. Cleaner HPLC chromatograms were obtained for milk samples submitted to the MISPE protocol in comparison to the solid phase extraction using the NIP or modified octadecyl silica (C18). Recoveries varying from 96.6 up to 102.2% for milk samples spiked with cholesterol were achieved, thus ensuring the accuracy of the proposed method.

Diagnóstico del síndrome Smith-Lemli-Opitz por cromatografía de capa fina / Smith-Lemli-Opitz syndrome diagnosis through thin film chromatography

El síndrome Smith-Lemli-Opitz es un error congénito del metabolismo del colesterol causante de malformaciones y retraso mental. El diagnóstico bioquímico consiste en la demostración del incremento del 7-dehidrocolesterol por cromatografía de gases/espectrometría de masa, tecnología que no siempre está disponible en los países en vías de desarrollo. Se propone un método de diagnóstico basado en la determinación cualitativa de esteroles por cromatografía de capa fina. El estudio en personas sanas no detectó la presencia del 7-dehidrocolesterol. En 21 pacientes con características clínicas de Smith-Lemli-Opitz se hicieron estudios por cromatografía de capa fina y por cromatografía de gases/espectrometría de masa y se diagnosticaron 10 pacientes, hubo una coincidencia total de los resultados por ambos métodos. En otros cuatro pacientes positivos por cromatografía de capa fina se identificaron las dos mutaciones causantes de la enfermedad. Los resultados avalan la utilidad del método propuesto para el diagnóstico del síndrome Smith-Lemli-Opitz…(AU)

Smith-Lemli-Opitz syndrome: clinical and biochemical findings in Brazilian patients

Smith-Lemli-Opitz syndrome (SLOS) or RSH syndrome comprises multiple congenital anomalies and mental retardation. The underlying defect is a deficiency in the activity of delta7-sterol reductase, which decreases cholesterol and increases 7-dehydrocholesterol (7-DHC) levels. Our aim was to identify and evaluate the frequency of SLOS manifestations in a group of Brazilian patients. Based on our own data and those reported previously, we present a simple method that allows the estimation of probabilities favoring the diagnosis of SLOS. We evaluated 30 patients clinically and determined their plasma levels of cholesterol and 7-dehydrocholesterol. In 11 patients, the diagnosis was confirmed by ultraviolet spectrophotometry (UV). Of 19 patients with normal laboratory results, 17 showed a high probability favoring the diagnosis of SLOS. The most significant signs and symptoms observed in over 2/3 of the biochemically confirmed cases were mental retardation (10/11), delayed neuropsychomotor development (10/11), syndactyly of 2nd/3rd toes (10/11), and craniofacial anomalies including microcephaly (11/11), incompletely rotated ears (8/11), palpebral ptosis (10/11), anteverted nostrils (10/11), and micrognathia (9/11). Genital anomalies were found in all male patients (6/6).

Characterization and properties of cholesterol desaturases from the ciliate Tetrahymena thermophila.

Live Tetrahymena thermophila transforms exogenous cholesterol into 7,22-bis, dehydrocholesterol (DHC) by desaturation at positions C7(8) and C22(23) of the cholesterol moiety. In this first report on expression, isolation, characterization, and reconstitution of Tetrahymena's cholesterol desaturases in cell-free extracts, we describe conditions for increasing the expression of both desaturases based on the addition of specific sterols to the culture medium. Reactions performed in vitro, with isolated microsomes, yield only the mono-unsaturated derivatives, 7-DHC and/or 22-DHC. However, selectivity towards one product can be improved with the addition of specific compounds: beta-mercaptoethanol inhibited C22(23) desaturase activity completely, while ethanol selectively increased this activity. Detergent-solubilized microsomes showed no desaturase activity, but partial restoration could be achieved with addition of dilauroyl-phosphatidylcholine liposomes (25%). Both cholesterol desaturases require molecular oxygen and cytochrome b(5). NADH or NADPH can serve as reduced cofactors, albeit with different efficiency, delivered by reductases present in the microsomal fraction. Azide and cyanide, but not azole compounds, inhibited these desaturases, suggesting a key role for cytochrome b(5) in these reactions.

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

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 3 -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 l 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.

Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light.

Solanum glaucophyllum contains the calciotropic hormone 1, 25-dihydroxy-vitamin D3 (1,25(OH)2D3). The metabolic pathway leading to the formation of 1,25(OH)2D3 in the plant is largely unknown. Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D3 and its metabolites. To investigate the requirement for light, S. glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and high-performance liquid chromatography. HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were detected. The presence of 1,25(OH)2D3 was also evidenced by [3H]1,25(OH)2D3 competitive binding analysis using the chick hormone intestinal receptor. Furthermore, 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were unequivocally identified by mass spectrometry. Incubation of control samples of 7-dehydrocholesterol under the same conditions as S. glaucophyllum cultures did not result in vitamin D3 formation, excluding the influence of light in these experiments. The results suggest that a synthetic route of vitamin D3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro.

Solar ultraviolet B radiation and photoproduction of vitamin D3 in central and southern areas of Argentina.

The incidence of nutritional rickets in the southern part of Argentina is 8-12 times higher than in the rest of the country. Winter 25(OH)D serum levels in normal population of southern areas are lower than in central and northern areas. To elucidate these differences, we compared the photoconversion of provitamin D3 (7-DHC) to previtamin D3 in two cities: Ushuaia (latitude 55 degrees S) and Buenos Aires (34 degrees S). Ampules containing 7-DHC were exposed to sunlight one day in the middle of each month either from 10:30 a.m. to 2:30 p.m. or from 8:00 a.m. to 5:00 p.m. The percentages of photoproducts formed were determined by high performance liquid chromatography (HPLC). Previous studies have proved that this is a valid model to assess "in vitro" the photoproduction of vitamin D3 in human skin. Previtamin D3 + vitamin D3 formed in Ushuaia were less (p < 0.02) than those found in Buenos Aires during all seasons: summer, (X +/- SEM) 6.4 +/- 0.8% vs. 13.2 +/- 1.8%; autumn, 1.2 +/- 0.7% vs. 6.3 +/- 1.3%; winter, 0.8 +/- 0.7% vs. 3.6 +/- 0.7%; spring, 3.4 +/- 0.5% vs. 9.1 +/- 1.1%. The photoproducts produced from 10:30 a.m. to 2:30 p.m. were similar for each month and latitude to those formed when the ampules were exposed from 8:00 a.m. to 5:00 p.m. We conclude that in Ushuaia there is a prolonged "vitamin D winter" during which cutaneous synthesis of vitamin D is absent, leading to lower serum values of 25(OH)D and contributing to the higher incidence of rickets.