G.S. Tint

Fetal Smith-Lemli-Opitz syndrome can be detected accurately and reliably by measuring amniotic fluid dehydrocholesterols

The Smith–Lemli–Opitz syndrome, characterized by limb, face and organ abnormalities, and mental retardation, is caused by an inherited block in the step of cholesterol biosynthesis in which the Δ7 double bond of 7‐dehydrocholesterol is reduced. It is diagnosed by the presence of markedly elevated levels of 7‐dehydrocholesterol and 8‐dehydrocholesterol in plasma and tissue. We measured amniotic fluid sterols in 15 pregnancies in 13 women who had previously carried an affected fetus. Cholesterol, 7–dehydrocholesterol and 8‐dehydrocholesterol concentrations averaged 18±3, 9·8±2·9 and 5·0±1·7 μg/ml, respectively, in seven pregnancies with an affected fetus or child. In contrast, these levels were 19±3, 0·05±0·01 and <0·005 μg/ml, respectively, in eight increasedrisk pregnancies with normal outcomes and 16±2, 0·07±0·01 and <0·005 μg/ml in normal controls. 7‐dehydrocholesterol concentrations, 2·2–26 and 0·05–0·10 μg/ml in pregnancies with an affected and unaffected fetus, respectively, did not overlap. Thus, abnormally elevated amniotic fluid dehydrocholesterol concentrations are an accurate predictor of fetal Smith–Lemli–Opitz syndrome. A false‐positive or a false‐negative result is highly unlikely.

The Smith—Lemli—Opitz Syndrome: A Potentially Fatal Birth Defect Caused by a Block in the Last Enzymatic Step in Cholesterol Biosynthesis

The Smith-Lemli-Opitz or “RSH” syndrome, first described in 1964 (Smith et al., 1964), is a severely debilitating and frequently fatal birth defect syndrome with an autosomal recessive mode of inheritance (Dellaire, 1969). Original estimates suggested an incidence of one in 20,000 births (Lowry and Yong, 1980) and a probable carrier frequency of about 1% (Chasalow, 1985). However, newer studies from the Czech Republic may place its true prevalence closer to 1 in 9,000, with as many as one in 45–50 inviduals carrying the defect (Opitz et al., 1997). Although all of the above estimates were made in case that were not biochemically confirmed, if the most recent observations prove to be correct, then the syndrome is as least as common as medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (Zaideh et al., 1995) and may well be the fourth most common recessively inherited disorder in Caucasians following cystic fibrosis, phenylketonuria, and hemochromatosis. Until the discovery in 1993 of the cholesterol biosynthesis defect in children with the syndrome (Irons et al., 1993; Tint et al., 1993; Tint et al., 1994), its cause was entirely unknown (Gorlin et al., 1990).

Comparative effects of lovastatin and chenodeoxycholic acid on plasma cholestanol levels and abnormal bile acid metabolism in cerebrotendinous xanthomatosis

We investigated the effect of the hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin and the primary bile acid chenodeoxycholic acid (CDCA) on plasma sterol and bile alcohol concentrations and the excretion of bile alcohols in urine in a 38-year-old homozygote with cerebrotendinous xanthomatosis (CTX). Untreated, plasma cholesterol concentrations were less than normal (171 +/- 5 v 185 +/- 3 mg/dL, P < .05) while plasma cholestanol levels were more than 20 times higher than the control mean (2.26 +/- 0.17 v 0.1 +/- 0.1 mg/dL, P < .0001). Plasma and urinary bile alcohol concentrations were markedly increased (12.6 +/- 0.6 and 154 micrograms/mL, respectively, v trace amounts in controls), with the ratio of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 25-tetrol to 5 beta-cholestane, 3 alpha,7 alpha,12 alpha,23 (22 and 24),25-pentols being 1.6 in plasma and reversed to 0.15 in urine. Treatment with lovastatin (40 mg/d) reduced plasma cholesterol concentrations 13%, but failed to decrease plasma cholestanol or bile alcohol levels. Abundant amounts of bile alcohols continued to be excreted in urine. In contrast, CDCA (750 mg/d) inhibited abnormal bile acid synthesis, as evidence by a 17-fold decrease in total bile alcohol levels in plasma and a 29-fold decrease in urine and the virtual elimination of cholic acid and deoxycholic acid from the bile. Plasma cholestanol concentrations also decreased 85%, but cholesterol levels increased 14%. The combination of CDCA with lovastatin did not improve plasma cholestanol or bile alcohol concentrations compared with CDCA treatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Lithium hydroxide/aqueous methanol: mild reagent for the hydrolysis of bile acid methyl esters

An efficient and convenient procedure for the hydrolysis of bile acid methyl esters is described. This is achieved by the addition of aqueous lithium hydroxide in methanol/dioxane (or tetrahydrofuran) at room temperature. Under these conditions, the formates as well as the acetate derivatives were also hydrolyzed, and the desired bile acids were isolated in 86 to 94% yield.

p-Toluenesulfonic acid/methanol: mild reagent for the preparation of bile acid methyl esters.

An improved method for the preparation of bile acid methyl esters is described. This is achieved by the addition of catalytic amounts of p-toluenesulfonic acid in a solution of bile acid in methanol. Advantages of this procedure over conventional methods include (1) use of a mild solid acid catalyst which prevents the formation of undesirable byproducts, (2) isolation of a solid product of high purity and (3) utilization of a relatively safe reagent in comparison to other methods involving diazomethane, hydrochloric acid or sulfuric acid.

Developmental sensitivity of associative learning to cholesterol synthesis inhibitors.

Patients with Smith-Lemli-Opitz syndrome, a genetic disorder associated with severe mental retardation, are unable to convert 7-dehydrocholesterol to cholesterol. Treatment of rats with agents that block cholesterol synthesis produces a sterol profile reminiscent of Smith-Lemli-Opitz patients i.e., low levels of cholesterol accompanied by the appearance of its immediate precursor 7-dehydrocholesterol. In previous work, chronic inhibition of cholesterol synthesis in just-weaned rats impaired acquisition of the classically conditioned eyeblink response. The present study had two primary goals--(1) to determine whether the learning impairment depended on the age in which treatment was initiated; and (2) to determine whether the deficit was associative or due to performance factors. Consistent with earlier work, acquisition of the eyeblink conditioned response was impaired when the 30-day treatment was initiated on postnatal day (PND) 21. Reactivity to acoustic stimuli and to eyelid stimulation were normal, suggesting that the learning impairment was associative in nature. The learning impairment was transitory; acquisition was normal when evaluated 30 days after the cessation of treatment. When treatment was initiated 30 days after weaning (PND 51), acquisition of the eyeblink response was normal. However, brain sterols of young adult rats were less affected than those of just-weaned rats. Thus, there is a developmental sensitivity to cholesterol synthesis blocking agents both in terms of their effects on brain sterols and new motor learning.

Identification of 5α-stanols in patients with sitosterolemia and xanthomatosis: stereochemistry of the protonolysis of steroidal organoboranes.☆

Plasma and fecal sterols of patients who exhibited tendon xanthomas with normal plasma cholesterol levels were studied. Plasma levels were (mg/dl mean +/- SD): cholesterol 232 +/- 36; cholestanol 5.9 +/- 2.1 (normal less than 0.6); sitosterol 18 +/- 5.6 (normal less than 1.0); campesterol 11 +/- 3.3 (normal less than 1.0). Other sterols such as sitostanol and campestanol (the 5 alpha-dihydro derivatives of sitosterol and campesterol) were also present in large amounts. Examination of the feces from these subjects showed cholesterol, sitosterol, campesterol and their 5 beta-saturated derivatives. Presence of 5 alpha-stanols in the plasma, and their absence in the feces indicates that the 5 alpha-stanols are synthesized endogenously within the body rather than in the intestine by colonic bacteria. The absolute identification of the structures of these 5 alpha-stanols was elucidated via hydroboration of their corresponding unsaturated sterols coupled with protonolysis of the resultant organoboranes.

Preparation of [3β-3H] labeled bile acids and bile alcohols ☆

[3beta-3H]-bile acids and bile alcohols may be useful for metabolic studies in man and animals because the 3-position is invulnerable to bacterial attack. A number of tritium labeled bile acids and bile alcohols were prepared by selective oxidation of the hydroxyl group at carbon-3 followed by reduction with NaBT4. In each case, the bile acids and bile alcohols epimeric at carbon-3 were resolved by analytical and preparative thin-layer chromatography and characterized by gasliquid chromatography. The average yield was 60--65% and specific activities of the final products were in the range of 7.4 x 10(7) dpm/mg.

Synthesis of biological precursors of cholic acid

This paper describes a new and convenient procedure for the synthesis of 5beta-cholestane-3alpha,7alpha,12alpha,24-tetrol (24R and 24 S) and 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol starting from 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol. Dehydration of the 25-hydroxytetrol with glacial acetic acid and acetic anhydride yielded a mixture of 5beta-cholest-24-ene-3alpha,7alpha,12alpha-triol and the corresponding delta25 compound. Hydroboration and oxidation of the mixture of delta24 and delta25 unsaturated bile alcohols resulted in the formation of 5beta-cholestane-3alpha,7alpha,12alpha,24epsilon-tetrol and 5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol. In addition, smaller amounts of 5beta-cholestane-3alpha,7alpha,12alpha,23epsilon-tetrol and 5beta-cholestane-3alpha,7alpha,12alpha-triol were also obtained. The bile alcohols epimeric at C-24 were resolved by analytical and preparative TLC, characterized by gas-liquid chromatography and mass-spectrometry. Tentatively assignments of the 24R and 24S configuration was made on the basis of molecular rotation differences. These compounds will be useful for biological studies of cholic acid biosynthesis.

Evidence for the early reduction of the 24,25 double bond in the conversion of lanosterol to cholesterol in cerebrotendinous xanthomatosis

The metabolism of lanosterol and 24,25-dihydrolanosterol (DL) was examined in a patient with cerebrotendinous xanthomatosis after intravenous pulse labeling with a mixture of DL-2-14C and 3S,4S,3R,4R-(4-3H)mevalonate. Sterols were isolated from the feces and purified by silver nitrate thin-layer chromatography, and their identities were confirmed by gas-liquid chromatography and mass spectrometry. Their specific activities were then determined and plotted as a function of time. These isotope ratio measurements and specific activity decay curves were consistent with 24,25-dihydrolanosterol and delta7-cholestenol being intermediates in the synthesis of cholesterol from mevalonate and lanosterol, and they suggested that reduction of the lanosterol side chain may occur as an early step in the synthesis of cholesterol. These results are in contrast to the results reported after the administration of triparanol, a delta24-reductase inhibitor.