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Sterol balance in the Smith-Lemli-Opitz syndrome: reduction in whole body cholesterol synthesis and normal bile acid production

2000; Elsevier BV; Volume: 41; Issue: 9 Linguagem: Inglês

10.1016/s0022-2275(20)33456-8

1539-7262

Robert D. Steiner, Leesa M. Linck, Donna P Flavell, Don S. Lin, William E. Connor,

Lipid metabolism and biosynthesis

The Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/mental retardation syndrome caused by a deficiency of the enzyme 7-dehydrocholesterol Δ7-reductase. This enzyme converts 7-dehydrocholesterol (7-DHC) to cholesterol in the last step in cholesterol biosynthesis. The pathology of this condition may result from two different factors: the deficiency of cholesterol itself and/or the accumulation of precursor sterols such as 7-DHC. Although cholesterol synthesis is defective in cultured SLOS cells, to date there has been no evidence of decreased whole body cholesterol synthesis in SLOS and only incomplete information on the synthesis of 7-DHC and bile acids. In this first report of the sterol balance in SLOS, we measured the synthesis of cholesterol, other sterols, and bile acids in eight SLOS subjects and six normal children. The diets were very low in cholesterol content and precisely controlled. Cholesterol synthesis in SLOS subjects was significantly reduced when compared with control subjects (8.6 vs. 19.6 mg/kg per day, respectively, P < 0.002). Cholesterol precursors 7-DHC, 8-DHC, and 19-nor-cholestatrienol were synthesized in SLOS subjects (7-DHC synthesis was 1.66 ± 1.15 mg/kg per day), but not in control subjects. Total sterol synthesis was also reduced in SLOS subjects (12 vs. 20 mg/kg per day, P < 0.022). Bile acid synthesis in SLOS subjects (3.5 mg/kg per day) did not differ significantly from control subjects (4.6 mg/kg per day) and was within the range reported previously in normals. Normal primary and secondary bile acids were identified. This study provides direct evidence that whole body cholesterol synthesis is reduced in patients with SLOS and that the synthesis of 7-DHC and other cholesterol precursors is profoundly increased. It is also the first reported measure of daily bile acid synthesis in SLOS and provides evidence that bile acid supplementation is not likely to be necessary for treatment. These sterol balance studies provide basic information about the biochemical defect in SLOS and strengthen the rationale for the use of dietary cholesterol in its treatment. —Steiner, R. D., L. M. Linck, D. P. Flavell, D. S. Lin, and W. E. Connor. Sterol balance in the Smith-Lemli-Opitz syndrome: reduction in whole body cholesterol synthesis and normal bile acid production. J. Lipid Res. 2000. 41: 1437–1447. The Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/mental retardation syndrome caused by a deficiency of the enzyme 7-dehydrocholesterol Δ7-reductase. This enzyme converts 7-dehydrocholesterol (7-DHC) to cholesterol in the last step in cholesterol biosynthesis. The pathology of this condition may result from two different factors: the deficiency of cholesterol itself and/or the accumulation of precursor sterols such as 7-DHC. Although cholesterol synthesis is defective in cultured SLOS cells, to date there has been no evidence of decreased whole body cholesterol synthesis in SLOS and only incomplete information on the synthesis of 7-DHC and bile acids. In this first report of the sterol balance in SLOS, we measured the synthesis of cholesterol, other sterols, and bile acids in eight SLOS subjects and six normal children. The diets were very low in cholesterol content and precisely controlled. Cholesterol synthesis in SLOS subjects was significantly reduced when compared with control subjects (8.6 vs. 19.6 mg/kg per day, respectively, P < 0.002). Cholesterol precursors 7-DHC, 8-DHC, and 19-nor-cholestatrienol were synthesized in SLOS subjects (7-DHC synthesis was 1.66 ± 1.15 mg/kg per day), but not in control subjects. Total sterol synthesis was also reduced in SLOS subjects (12 vs. 20 mg/kg per day, P < 0.022). Bile acid synthesis in SLOS subjects (3.5 mg/kg per day) did not differ significantly from control subjects (4.6 mg/kg per day) and was within the range reported previously in normals. Normal primary and secondary bile acids were identified. This study provides direct evidence that whole body cholesterol synthesis is reduced in patients with SLOS and that the synthesis of 7-DHC and other cholesterol precursors is profoundly increased. It is also the first reported measure of daily bile acid synthesis in SLOS and provides evidence that bile acid supplementation is not likely to be necessary for treatment. These sterol balance studies provide basic information about the biochemical defect in SLOS and strengthen the rationale for the use of dietary cholesterol in its treatment. —Steiner, R. D., L. M. Linck, D. P. Flavell, D. S. Lin, and W. E. Connor. Sterol balance in the Smith-Lemli-Opitz syndrome: reduction in whole body cholesterol synthesis and normal bile acid production. J. Lipid Res. 2000. 41: 1437–1447. The Smith-Lemli-Opitz syndrome (SLOS) (1Smith D. Lemli L. Opitz J. A newly recognized syndrome of multiple congenital anomalies.J. Pediatr. 1964; 64: 210-217Google Scholar) is an autosomal recessive disorder. It is characterized by microcephaly, cleft palate, mental retardation, growth retardation, dysmorphic facies, limb abnormalities (especially syndactyly of the toes), genital disorders, endocrine mal-function, cataracts, and heart and kidney malformations (1Smith D. Lemli L. Opitz J. A newly recognized syndrome of multiple congenital anomalies.J. Pediatr. 1964; 64: 210-217Google Scholar, 2Curry C.J. Carey J.C. Holland J.S. Chopra D. Fineman R. Golabi M. Sherman S. Pagon R.A. Allanson J. Shulman S. Barr M. McGravey V. Dabiri C. Schimke N. Ives E. Hall B. Smith-Lemli-Opitz syndrome-type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality.Am. J. Med. Genet. 1987; 26: 45-57Google Scholar, 3Pober B. Smith-Lemli-Opitz syndrome.in: Buyse M. Birth Defects Encyclopedia. Blackwell Scientific, Dover, MA1990: 1570-1572Google Scholar, 4Opitz J.M. Penchaszadeh V.B. Holt M.C. Spano L.M. Smith-Lemli-Opitz (RSH) syndrome bibliography.Am. J. Med. Genet. 1987; 28: 745-750Google Scholar, 5Chasalow F.I. Blethen S.L. Taysi K. Possible abnormalities of steroid secretion in children with Smith-Lemli-Opitz syndrome and their parents.Steroids. 1985; 46: 827-843Google Scholar, 6Natowicz M.R. Evans J.E. Abnormal bile acids in the Smith-Lemli-Opitz syndrome.Am. J. Med. Genet. 1994; 50: 364-367Google Scholar, 7Cunniff C. Kratz L.E. Moser A. Natowicz M.R. Kelley R.I. Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism.Am. J. Med. Genet. 1997; 68: 263-269Google Scholar, 8Ryan A.K. Bartlett K. Clayton P. Eaton S. Mills L. Donnai D. Winter R.M. Burn J. Smith-Lemli-Opitz syndrome: a variable clinical and biochemical phenotype.J. Med. Genet. 1998; 35: 558-565Google Scholar). Affected individuals may have virtually all these features or may be quite mildly affected with only growth impairment, subtle dysmorphic facial features, toe syndactyly, and learning disability. Heterozygotes (carriers) have no discernible phenotype. No proven therapy is available. SLOS is estimated to occur in 1 in 20,000 births, yielding an estimated carrier frequency of 1 to 2%, making it one of the most common autosomal recessive disorders (4Opitz J.M. Penchaszadeh V.B. Holt M.C. Spano L.M. Smith-Lemli-Opitz (RSH) syndrome bibliography.Am. J. Med. Genet. 1987; 28: 745-750Google Scholar, 8Ryan A.K. Bartlett K. Clayton P. Eaton S. Mills L. Donnai D. Winter R.M. Burn J. Smith-Lemli-Opitz syndrome: a variable clinical and biochemical phenotype.J. Med. Genet. 1998; 35: 558-565Google Scholar). Tint and colleagues found reduced cholesterol and elevated 7-dehydrocholesterol (7-DHC) levels in the plasma and tissues of these patients and postulated that SLOS resulted from a defect in cholesterol and bile acid biosynthesis (9Tint G.S. Irons M. Elias E.R. Batta A.K. Frieden R. Chen T.S. Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome.N. Engl. J. Med. 1994; 330: 107-113Google Scholar, 10Irons M. Elias E.R. Salen G. Tint G.S. Batta A.K. Defective cholesterol biosynthesis in Smith-Lemli-Opitz syndrome [letter].Lancet. 1993; 341: 1414Google Scholar, 11Tint G.S. Cholesterol defect in Smith-Lemli-Opitz syndrome [letter].Am. J. Med. Genet. 1993; 47: 573-574Google Scholar, 12Tint G.S. Seller M. Hughes-Benzie R. Batta A.K. Shefer S. Genest D. Irons M. Elias E. Salen G. Markedly increased tissue concentrations of 7-dehydrocholesterol combined with low levels of cholesterol are characteristic of the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1995; 36: 89-95Google Scholar). Besides 7-DHC, other sterol derivatives (e.g., 8-dehydrocholesterol) have been found in the plasma of these patients (13Batta A.K. Tint G.S. Shefer S. Abuelo D. Salen G. Identification of 8-dehydrocholesterol (cholesta-5,8-dien-3 betaol) in patients with Smith-Lemli-Opitz syndrome.J. Lipid Res. 1995; 36: 705-713Google Scholar). Deficiency of the human sterol Δ7-reductase enzyme (7-dehydrocholesterol Δ7-reductase; EC 1.3.1.21) was subsequently shown in hepatocytes and fibroblasts from affected individuals (14Honda A. Tint G.S. Salen G. Batta A.K. Chen T.S. Shefer S. Defective conversion of 7-dehydrocholesterol to cholesterol in cultured skin fibroblasts from Smith-Lemli-Opitz syndrome homozygotes.J. Lipid Res. 1995; 36: 1595-1601Google Scholar, 15Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol-delta 7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar). This enzyme converts 7-DHC to cholesterol in the final step of cholesterol biosynthesis. With our collaborator, F. D. Porter, and colleagues, we and others have found that mutations in the 7-dehydrocholesterol Δ7-reductase gene (DHCR7) cause SLOS (16Fitzky B.U. Witsch-Baumgartner M. Erdel M. Lee J.N. Paik Y.K. Glossmann H. Utermann G. Moebius F.F. Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome.Proc. Natl. Acad. Sci. USA. 1998; 95: 8181-8186Google Scholar, 17Wassif C.A. Maslen C. Kachilele-Linjewile S. Lin D. Linck L.M. Connor W.E. Steiner R.D. Porter F.D. Mutations in the human sterol delta7-reductase gene at 11q12–13 cause Smith-Lemli-Opitz syndrome.Am. J. Hum. Genet. 1998; 63: 55-62Google Scholar, 18Waterham H.R. Wijburg F.A. Hennekam R.C. Vreken P. Poll-The B.T. Dorland L. Duran M. Jira P.E. Smeitink J.A. Wevers R.A. Wanders R.J. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene.Am. J. Hum. Genet. 1998; 63: 329-338Google Scholar). The clinical manifestations of SLOS may result from cholesterol deficiency or from the toxicity of precursor sterols, particularly 7-DHC (normally absent or detected in only trace quantities in plasma). Cells use cholesterol for membrane synthesis and as a precursor for steroid hormones and bile acids. Cholesterol is also needed for autoprocessing (activation) of Sonic hedgehog (Shh), an important protein in the early limb patterning and craniofacial development in the human embryo (19Porter J.A. von Kessler D.P. Ekker S.C. Young K.E. Lee J.J. Moses K. Beachy P.A. The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.Nature. 1995; 374: 363-366Scopus (436) Google Scholar, 20Porter J. Porter K. Young P. Cholesterol modification of hedgehog signaling proteins in animal development.Science. 1996; 274: 255-259Google Scholar, 21Porter J.A. Ekker S.C. Park W.J. von Kessler D.P. Young K.E. Chen C.H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain.Cell. 1996; 86: 21-34Google Scholar). Abnormal Shh activation and/or signaling may explain the malformations in SLOS (22Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Teratogen-mediated inhibition of target tissue response to Shh signaling.Science. 1998; 280: 1603-1607Google Scholar). 7-DHC likely does play a role in the pathogenesis of SLOS. 7-DHC impairs learning in a rat model of SLOS, where rats are treated with an inhibitor of 7-dehydrocholesterol Δ7-reductase, BM 15.766. The learning impairment was prevented by administration of supplemental cholesterol, which lowered the 7-DHC content (but did not raise cholesterol content) in rat brain (23Xu G.R. Servatius R.J. Shefer S. Tint G.S. O'Brien W.T. Batta A.K. Salen G. Relationship between abnormal cholesterol synthesis and retarded learning in rats.Metabolism. 1998; 47: 878-882Google Scholar). AY 9944 is another inhibitor of the 7-dehydrocholesterol Δ7-reductase enzyme. Oxidized derivatives of 7-DHC induce growth retardation and embryotoxicity in cultured AY 9944-treated rat embryos. Cholesterol supplementation ameliorated the growth retardation and morphologic abnormalities normally caused by the inhibitor in these embryos. Supplementation with 7-DHC, on the other hand, did not restore growth and, in fact, impaired the beneficial effects of cholesterol added simultaneously. Photooxidation of the 7-DHC-supplemented culture medium enhanced the embryotoxicity of 7-DHC (24Gaoua W. Chevy F. Roux C. Wolf C. Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1999; 40: 456-463Google Scholar). Supplemental dietary cholesterol may be beneficial for SLOS patients because they appear to have a cholesterol deficiency syndrome (25Connor W.E. A cholesterol deficiency syndrome in humans [editorial; comment].J. Clin. Invest. 1995; 95: 2Google Scholar, 26Irons M. Elias E.R. Tint G.S. Salen G. Frieden R. Buie T.M. Ampola M. Abnormal cholesterol metabolism in the Smith-Lemli-Opitz syndrome: report of clinical and biochemical findings in four patients and treatment in one patient.Am. J. Med. Genet. 1994; 50: 347-352Google Scholar, 27Nwokoro N.A. Mulvihill J.J. Cholesterol and bile acid replacement therapy in children and with Smith-Lemli-Opitz (Slo/Rsh) syndrome.Am. J. Med. Genet. 1997; 68: 315-321Google Scholar, 28Elias E.R. Irons M.B. Hurley A.D. Tint G.S. Salen G. Clinical effects of cholesterol supplementation in six patients with the Smith-Lemli-Opitz syndrome (SLOS).Am. J. Med. Genet. 1997; 68: 305-310Google Scholar). In addition, production of precursors of cholesterol synthesis that might contribute to the SLOS phenotype might also be inhibited by cholesterol supplementation due to inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Basic information about the synthesis of cholesterol, precursor sterols, and bile acids and methods for evaluating the effects of cholesterol supplementation on SLOS subjects are needed to understand more fully the biochemical defects before truly rational and effective therapy can be devised. As a prelude to development of a rational therapy for SLOS, we sought in this initial investigation to measure whole body cholesterol and sterol precursor synthesis as well as bile acid synthesis under steady state conditions in subjects with SLOS and normal age-matched control subjects. Such measurements can best be accomplished by the sterol balance technique. We hypothesized that cholesterol and bile acid synthesis in SLOS subjects would be markedly reduced compared with control subjects because of the enzymatic block in cholesterol synthesis, but that total sterol synthesis would be normal. We also hypothesized that synthesis of cholesterol precursors could be measured by the sterol balance technique. Data on cholesterol and bile acid synthesis in SLOS subjects will serve as a basis for optimizing the dose of cholesterol (and possibly bile acids) needed for potential therapy. On the basis of our current study, we report that whole body cholesterol synthesis is reduced in SLOS, that total sterol synthesis is reduced in SLOS subjects, and that bile acid synthesis in SLOS does not differ significantly from that in control subjects. These studies were approved by the Oregon Health Sciences University (OHSU, Portland, OR) Investigational Review Board and for all subjects studied the parents gave informed consent. Eight subjects with SLOS were enrolled. Their ages, gender, weight, and cholesterol, 7-DHC, 8-DHC, 19-nor-cholestatrienol, and total sterol levels after consuming very low cholesterol diets are listed in Table 1. Low plasma cholesterol levels and the characteristic accumulation of 7-DHC and other cholesterol precursors in plasma were observed. The mean plasma cholesterol level was 84 mg/dl and means of the abnormal sterols were 10.1, 6.9, and 1.4 mg/dl for 7-DHC, 8-DHC, and 19-nor-cholestatrienol, respectively. Three of the SLOS subjects had cholesterol levels greater than 100 mg/dl, suggesting that the diagnosis might have been missed if biochemical testing included only cholesterol level rather than sterol profile. All the subjects have SLOS type I; none would be classified as the more severe SLOS type II. Not all subjects had severity scores calculated, but the least severely affected patient has a severity score of 11. Control subjects ranged in age from 1 to 5 years. Control subjects 3–6 were studied as inpatients and are siblings of four SLOS subjects. The mean age of control subjects was 2.8 years, and the mean weight was 16 kg (range, 11.1–26.7 kg). We attempted to use age-matched control subjects but were unsuccessful in studying control subjects as young (6 weeks) as the youngest SLOS subject or as old (13 years) as the oldest SLOS subject. Stools in young children are often too runny to use in sterol balance studies and older children are often unwilling to collect their stools.TABLE 1.Characteristics and plasma sterol levels of SLOS subjects under conditions of very low cholesterol dietsSubjectSexAgeWeightCholesterol7-DHC8-DHC19-nora19-nor-Cholestatrienol.Totalkgmg/dlmg/dlmg/dlmg/dlmg/dl1M6 wk3.8575.59.404.501.290.62M7 mo5.3788.313.212.92.1116.53M1 yr5.5452.811.07.901.973.64F2.25 yr9.4101.62.302.500.3106.75F2.5 yr11.459.511.69.701.582.36F2.75 yr8.9087.610.53.600.8102.57F3.5 yr11.3100.99.402.300.8113.48M13 yr61.8104.513.611.82.6132.5Means ± SD83.8 ± 19.610.1 ± 3.56.9 ± 4.21.4 ± 0.8102.3 ± 19.3a 19-nor-Cholestatrienol. Open table in a new tab SLOS subjects and control subjects 3–6 were admitted to the OHSU General Clinical Research Center (GCRC) for 1-week periods. Instructions were given for an essentially cholesterol-free diet to be fed at home for three or more weeks prior to admission to the GCRC. This was easily accomplished in most cases because many of the infants were receiving exclusively infant formula containing cholesterol concentrations of only 10.5–35.7 mg/1,000 ml. During each admission a very low cholesterol (essentially cholesterol-free) diet was fed. The study diet was fed for at least 3 weeks total to allow for stabilization and steady state conditions. The subjects were studied in the GCRC under metabolic ward conditions. GCRC dieticians and cooks prepared the specialized diets and nurses collected patient samples at baseline and during the study periods. The food intake for infants consisted of commercially available infant formula feedings plus pureed cereals, fruits, and vegetables in which protein contributed 15–20%, fat 20–30%, and carbohydrate 45–55% of the total calorie intake and cholesterol content was low. The diets were provided with precise cholesterol content known and controlled. Older children were fed mixed general foods with the same caloric distribution (29Connor W. Stone D. Hodges R. The interrelated effects of dietary cholesterol and fat upon human serum lipid levels.J. Clin. Invest. 1964; 43: 1691-1696Google Scholar). The dietary prescriptions met the daily recommended allowances of the National Research Council. Some SLOS subjects were tube fed because of sucking and swallowing difficulties, but the same principles were applied to the diets of those individuals. The mothers of the children were given dietinstruction and were asked to keep intake records. For inpatient studies, food and formula were weighed prior to being ser ved to the subjects and refused food and formula were returned to the metabolic kitchen to be reweighed to determine actual intake. Control subjects 1 and 2 were unrelated healthy infants and children (the SLOS subjects were all infants and children). These control subjects were studied as outpatients, using published guidelines (30Cressey D.E. Kris-Etherton P.M. Zavoral J.H. Experimental note: sterol balance study conducted in the home.J. Am. Diet. Assoc. 1978; 73: 543-545Google Scholar, 31Nestel P.J. Poyser A. Boulton T.J. Changes in cholesterol metabolism in infants in response to dietary cholesterol and fat.Am. J. Clin. Nutr. 1979; 32: 2177-2182Google Scholar) with our modifications below. Control subjects 3–6 were siblings of SLOS subjects and were studied as inpatients. Diet instruction was provided by a registered dietitian; both oral and written instructions were provided. The dietitian was in frequent contact, every 1–3 days, with the parents during the study, either by phone or actual meetings. A record was kept by the parent of the subjects' dietary intake. The nutrient content of the diet was calculated from manufacturer information and by using the Food Processor Plus Nutrient Analysis Program (version 7.02; ESHA Research, Salem, OR). We have developed a system for performing sterol balance in infants and children without having to use a metabolic frame. We have been able to scrape frozen stool from cloth diapers and measure sterols and bile acids in pooled stool samples. Toilet-trained subjects collected all stools and the stools were individually placed in labeled plastic bags and the samples frozen for later analysis. Where stool and urine were mixed, or when stool could not be scraped off the diapers accurately, the samples were discarded, and analysis of sterol balance was not accomplished. For outpatients, refrigerators were distributed for stool collections, and stools were brought in at the end of a 1-week period of collection. These stools were then processed exactly as the stools of the SLOS subjects. Some stool may be left on the diapers after scraping. This would lead to an underestimate of cholesterol, sterol, and bile acid synthesis. We tried to minimize this error by not including sterol balance studies where stools were too runny to allow easy scraping off of diapers. We have also performed a series of experiments determining how much stool (and therefore sterols) is left behind when scraping diapers by extracting stool remaining in diapers after scraping and quantitating. The amount left behind was minuscule and not enough to extract for analysis. Synthesis of cholesterol and other sterols was analyzed by the sterol balance technique in SLOS subjects and control subjects. In this study, we analyzed the fecal sterol excretion of eight SLOS subjects and six normal control subjects by the methods initially developed by Ahrens, Grundy, and Miettinen (32Grundy S. Ahrens Jr., E. Miettinen T. Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral sterols.J. Lipid Res. 1965; 6: 397-410Google Scholar, 33Miettinen T. Ahrens Jr., E. Grundy S. Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral sterols.J. Lipid Res. 1965; 6: 411-424Google Scholar) and modified by us (34Connor W.E. Witiak D.T. Stone D.B. Armstrong M.L. Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition.J. Clin. Invest. 1969; 48: 1363-1375Google Scholar, 35Connor W.E. Lin D.S. The intestinal absorption of dietary cholesterol by hypercholesterolemic (type II) and normocholesterolemic humans.J. Clin. Invest. 1974; 53: 1062-1070Google Scholar, 36Lin D.S. Connor W.E. The long term effects of dietary cholesterol upon the plasma lipids, lipoproteins, cholesterol absorption, and the sterol balance in man: the demonstration of feedback inhibition of cholesterol biosynthesis and increased bile acid excretion.J. Lipid Res. 1980; 21: 1042-1052Google Scholar, 37McMurry M.P. Connor W.E. Lin D.S. Cerqueira M.T. Connor S.L. The absorption of cholesterol and the sterol balance in the Tarahumara Indians of Mexico fed cholesterol-free and high cholesterol diets.Am. J. Clin. Nutr. 1985; 41: 1289-1298Google Scholar). The sterol balance technique is based on the concept that in the metabolic steady state, the input of sterol into the body (the intake of dietary cholesterol and the endogenous synthesis of cholesterol) is balanced by the output (fecal excretion of neutral sterols and bile acids). Therefore, whole body sterol synthesis can be estimated by subtracting intake from excretion (38Grundy S.M. Ahrens Jr., E.H. Measurements of cholesterol turnover, synthesis, and absorption in man, carried out by isotope kinetic and sterol balance methods.J. Lipid Res. 1969; 10: 91-107Google Scholar). In SLOS subjects, the sterol balance technique also provides an estimation of the synthesis of 7-DHC and other cholesterol precursor sterols by measurement of these individual sterols in stool samples. All these patients and control subjects were consuming a very low cholesterol diet. Seven-day stools of these subjects were pooled. Stools were homogenized with equal amounts of water. An aliquot was taken and frozen immediately. For analysis of fecal sterols, 0.5–1.0 g of fecal homogenate was weighed out and traces of [4-14C]cholesterol and [24-14C]deoxycholic acid were added to monitor the recover y. After mild saponification, the fecal neutral sterols were extracted from bile acids. The lipid extracts of fecal neutral sterols were then subjected to thin-layer chromatography (silica gel H TLC plate with solvent ether–heptane 55:45) to separate sterols from stanols plus stanones (bacterially modified products). Sterols and stanols, plus stanone, were extracted from the TLC plate. Trimethylsilyl ether (TMS) derivatives of these compounds were subjected to analysis by gas–liquid chromatography (GLC). Because of the complex nature of the fecal sterols of these infants, to obtain complete resolution of these compounds it is necessary to analyze these samples by GLC with nonpolar as well as polar columns. Therefore, the samples were first analyzed with a GLC equipped with a hydrogen flame ionization detector (3B gas chromatograph; Perkin-Elmer, Norwalk, CT) and containing a nonpolar 30-m SE-30 capillary column with 0.25-mm i.d. and 0.25-μm film thickness. The samples were also analyzed with a Perkin-Elmer gas chromatograph (model 8500) equipped with a polar 25-m CP-wax-57 capillary column (Chrompack-Varian, Walnut Creek, CA) with 0.32-mm i.d. and 0.25-μm film thickness. The temperatures of the columns were 260°C for the SE-30 column and 200°C for the CP-wax column. Helium was used as the carrier gas, and cholestane as an internal standard. After removing neutral sterols, the aqueous layer containing the bile acids was put in a pressure cooker at 15 psi to cleave conjugated bile acids. The free bile acids were then extracted with chloroform–methanol and methylated with diazomethane. Methyl esters of bile acids were further purified by TLC. The region including and between cholic acid and lithocholic acid on the plate was scraped. Bile acids were extracted from the plate and TMS derivatives made. The samples were run on the GLC equipped with the SE-30 columns. Bile acids were separated and quantified. Considering the possible loss of aalyzed compounds in these lengthy procedures, we added [4-14C]cholesterol and [24-14C]deoxycholic acid to the fecal homogenate to monitor the loss of neutral sterols and bile acids. The recovery of labeled cholesterol was 78.6 ± 9.9% for SLOS subjects and 79.7 ± 8.8% for control subjects. Recovery for bile acids was 93 ± 7.1% for SLOS subjects and 90.8 ± 8.5% for control subjects. We used the percent recovery to correct for loss of sample in the TLC step in the calculations of sterol and bile acid synthesis. From the daily excretion of fecal steroids (neutral sterols and bile acids) and the cholesterol intake, we measured the daily total sterol synthesis of these subjects by the balance technique (intake–total excretion). Because identification of the bile acids made from the noncholesterol compounds is problematic, precise calculation of the synthesis is not yet possible. Therefore, we assumed that the quantity of bile acid from these compounds was proportional to the quantity of their precursor neutral sterols in the stool. With this assumption, using the total bile acid excretion, we estimated the production of bile acid from these sterols. This estimate will yield an upper limit estimate of individual sterol and bile acid synthesis from precursor sterols because bile acids may not be made in great quantities from precursor sterols because dehydrocholesterols cannot likely be converted to bile acids via the 7α-hydroxylase pathway. The first step in bile acid synthesis is 7α hydroxylation. It is likely that the double bond at the 7-carbon of 7-DHC blocks this reaction. Some 7-DHC and other precursors may be converted to bile acids via the 27-hydroxylation pathway but the amount is likely to be small (39Honda A. Salen G. Shefer S. Batta A.K. Honda M. Xu G. Tint G.S. Matsuzaki Y. Shoda J. Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.J. Lipid Res. 1999; 40: 1520-1528Google Scholar). The estimates for sterol synthesis are likely to be close to the actual synthesis because the majority of these compounds are found in stool in the neutral sterol fraction rather than as bile acids, so that the contribution of bile a