Ashok K. Batta

Simultaneous quantitation of fatty acids, sterols and bile acids in human stool by capillary gas-liquid chromatography

A simple method for the simultaneous gas-liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.

Cholesterol and oxygenated cholesterol concentrations are markedly elevated in peripheral tissue but not in brain from mice with the Niemann–Pick type C phenotype

Niemann–Pick disease type C (NP-C) is a rare genetic disorder characterized by progressive neurodegeneration, frequent developmental delay and early death. Tissues of affected individuals accumulate large quantities of free cholesterol in lysosomes. Because cytotoxic oxygenated derivatives of cholesterol are known to form readily when cholesterol concentrations are elevated, we searched for these compounds in liver, kidney, spleen and brain from mice with the NP-C phenotype. In order of abundance, we identified 7α- and 7β-hydroxycholesterol, 5α,6α-epoxycholestan-3β-ol, 4β-hydroxycholesterol, cholest-4-en-3β,7α-diol and cholest-4-en-3β,6β-diol in most tissue samples. Cholesterol concentrations in affected mice were increased 3-fold in kidney and 7- to 8-fold in spleen and liver compared to controls (all p<0.001) but were unchanged in brain. Although oxysterol levels were markedly elevated in non-brain tissue, the oxysterol and cholesterol concentrations increased proportionally so that oxysterols expressed as percentage of total sterols were the same for all animals (0.34±0.19% averaged over all organs in affected animals vs 0.40±0.42% in control mice). In contrast to peripheral tissue, we could not detect any increase in either absolute or relative oxysterol levels in the brains of affected and control mice (49±61 vs 53±43 μg/g wet weight and 0.45±0.52 vs 0.47±0.37%, respectively). Thus, brain sterols are normal in NP-C mice and it is unlikely that an accumulation of cytotoxic oxygenated derivatives of cholesterol could account for the progressive neuropathology seen in the disease.

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7α-hydroxylase activities in the rat, guinea pig, and rabbit: Effects of cholesterol and bile acids

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

Regulation of bile acid synthesis by deoxycholic acid in the rat: Different effects on cholesterol 7α‐hydroxylase and sterol 27‐hydroxylase

We examined the effects of feeding deoxycholic acid (1% and 0.4% of diet), alone and in combination with ursodeoxycholic acid, on serum and biliary bile acid concentrations, hepatic morphology, and the activities and steady‐state messenger RNA (mRNA) levels of HMG‐CoA reductase and cholesterol 7α‐hydroxylase in the rat. Feeding 1% deoxycholic acid increased serum bile acid concentrations (cholestasis), produced portal traid inflammation, bile duct proliferation, and severe hepatocyte necrosis with nuclear pleomorphism. Hepatic demage was preventage when ursodeoxycholic acid (1%) was combined with the deoxycholic acid (1%), or when deoxycholic acid intake was reduced to 0.4%. HMG‐CoA reductase and cholesterol 7α‐hydroxylase activities were markedly inhibited (−56% and −55%, respectively) with either 1% or 0.4% deoxycholic acid. Ursodeoxycholic acid alone produced an insignificant decline in HMG‐CoA reductase and cholesterol 7α‐hydroxylase activities, and when combined with 1% deoxycholic acid did not lessen the inhibitory effect of the latter. Steady‐state mRNA levels increased 20‐fold for HMG‐CoA reductase and 53‐fold for cholesterol 7α‐hydroxylase in rats fed 1% deoxycholic acid. In contrast, 0.4% deoxycholic acid decreased HMG‐CoA reductase mRNA levels 76%, and cholesterol 7α‐hydroxylase mRNA levels 82%. Ursodeoxycholic acid alone did not affect HMG‐CoA reductase or cholesterol 7α‐hydroxylase steady‐state mRNA levels. Steady‐state mRNA levels and activities of sterol 27‐hydroxylase, a key enzyme in the alternative acidic pathway of bile acid synthesis, did not change with either high or low doses of deoxycholic acid. In conclusion, 1% deoxycholic acid induced hepatocyte destruction and regeneration associated with increased mRNA levels for HMG‐CoA reductase and cholesterol 7α‐hydroxylase, but significantly suppressed both enzyme activities. Thus, high‐dose deoxycholic acid uncouples HMG‐CoA reductase and cholesterol 7α‐hydroxylase mRNA levels from enzyme function. In contrast, lower‐dose deoxycholic acid (0.4%) inhibited both activities and mRNA levels of HMG‐CoA reductase and cholesterol 7α‐hydroxylase. Adding 1% ursodeoxycholic acid to 1% deoxycholic acid prevented the rise in mRNA levels but did not lessen the inhibitory effect of the latter. This inhibition occurred without change in hepatic histology, which suggests a regulatory role for deoxycholic acid that is independent of liver damage. Conversely, sterol 27‐hydroxylase activity and mRNA levels are not affected by deoxycholic acid treatments. (HEPATOLOGY 1995; 22:1215–1221.).

Relationship Between Abnormal Cholesterol Synthesis and Retarded Learning in Rats

We examined the relationship between brain sterol composition and associative learning (classical conditioning of the eyeblink response) in newly weaned rats fed BM 15.766 (BM) for 4 months. This compound inhibits 7-dehydrocholesterol-delta7-reductase, which catalyzes the conversion of 7-dehydrocholesterol to cholesterol, the last step in the synthetic pathway. As countertreatment, half of the BM-treated rats were fed 2% cholesterol during the last 2 months. With BM, cholesterol concentrations declined 91% in plasma, but with cholesterol feeding, the levels increased 50% compared with baseline values. 7-Dehydrocholesterol, which was not detected at baseline, increased to 55% of plasma sterols with BM but decreased to 5% of total plasma sterols when cholesterol was added. With BM, brain cholesterol levels decreased 60% and did not increase after cholesterol was added. However, 7-dehydrocholesterol, which comprised 39% of brain sterols with BM, decreased to 31% (P < .05) when cholesterol was fed. Hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity in the liver increased 2.2-fold with BM and declined 95% after adding cholesterol, but did not change in the brain. BM treatment for 4 months prevented learning of the conditioned eyeblink response as compared with controls. In contrast, BM-treated rats supplemented with cholesterol acquired the conditioned eyeblink response. Chronic inhibition of 7-dehydrocholesterol-delta7-reductase reduced cholesterol and increased 7-dehydrocholesterol levels in plasma and brain, and was associated with impaired learning. Cholesterol feeding corrected plasma and hepatic sterol levels and reduced brain 7-dehydrocholesterol concentrations to reestablish normal learning.

Sterol concentrations in cultured Smith-Lemli-Opitz syndrome skin fibroblasts: Diagnosis of a biochemically atypical case of the syndrome

The Smith-Lemli-Opitz syndrome is a common birth defect syndrome caused by a deficiency of 7-dehydrocholesterol delta 7-reductase, an essential enzyme in the biosynthesis of cholesterol. The syndrome can usually be diagnosed easily from the plasma markers of markedly elevated 7-dehydrocholesterol and reduced cholesterol concentrations. However, atypical cases with normal plasma levels of cholesterol with only moderately elevated 7-dehydrocholesterol have been reported. To establish a sensitive method for the biochemical diagnosis of the atypical cases of the syndrome, we measured sterol concentrations of cultured skin fibroblasts. 7-Dehydrocholesterol concentrations in patients fibroblasts grown in the presence of 10% fetal bovine serum were significantly higher than those in controls and parents (P < 0.0005), but they were not elevated proportionately as much as in plasma. To re-produce the accumulation of 7-dehydrocholesterol, the cells were exposed to delipidated medium to induce sterol biosynthesis. After 4 weeks, 7-dehydrocholesterol concentrations in patients fibroblasts increased from 2.8 +/- 0.3% to 34 +/- 3% of total sterols (cholesterol + 7-dehydrocholesterol + 8-dehydrocholesterol). The increase was also observed in fibroblasts from an atypical patient who has a normal plasma cholesterol level and a 7-dehydrocholesterol concentration of only 0.15 mg/dl. In contrast, cells from parents and controls accumulated very little 7-dehydrocholesterol (< 1% of total sterols). These results demonstrate that cultured fibroblasts exhibit abnormally high accumulation of 7-dehydrocholesterol after cells are exposed to delipidated medium not only in typical patients, but also in an atypical case. The present method is a sensitive procedure for the biochemical diagnosis of this syndrome.

Screening for abnormal cholesterol biosynthesis in the Smith‐Lemli‐Opitz syndrome: Rapid determination of plasma 7‐dehydrocholesterol by ultraviolet spectrometry

The Smith-Lemli-Opitz syndrome (SLOS) is a common condition caused by deficiency of 7-dehydrocholesterol delta 7-reductase. The syndrome can usually be diagnosed by demonstrating markedly increased plasma concentrations of the cholesterol precursor, 7-dehydrocholesterol. We describe a simple and rapid method for detection of plasma 7-dehydrocholesterol by use of ultraviolet (UV) spectrometry. Lipids were extracted from plasma by addition of ethanol and n-hexane, and the n-hexane phase was directly subjected to spectrometry. The absorption maxima characteristics of 7-dehydrocholesterol (lambda max 271, 282, and 294 nm) were observed in patients plasma but not in controls. For quantitative measurements, absorbance at 282 nm was used. Since this absorbance is the sum of the absorbance derived from 7-dehydrocholesterol and background absorbance, the concentrations of 7-dehydrocholesterol in various plasma samples were quantified by subtracting estimated background absorbance at 282 nm from observed absorbance at 282 nm. The results correlated well with total (free plus esterified) 7-dehydrocholesterol concentrations measured by gas-liquid chromatographic method. The UV spectrometric assay was sensitive enough to detect increased 7-dehydrocholesterol in cultured skin fibroblasts from patients grown in delipidated medium. The present method will make it possible to screen plasma or fibroblasts to detect the syndrome rapidly in general clinical laboratories.

Accurate detection of Smith–Lemli–Opitz syndrome carriers by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts

The activity of ergosterol Δ7-reductase (3β-hydroxysteroid Δ7-reductase) was measured in cultured skin fibroblasts from 7 controls, 10 Smith–Lemli–Opitz syndrome (SLOS) patients, and 10 parents (obligate carriers). The fibroblasts were exposed to delipidated medium supplemented with lovastatin for 24h and the enzyme activity was determined by incubating cell-free homogenate with ergosterol (ergosta-5,7,22-trien-3β-ol) and measuring the mass of brassicasterol (ergosta-5,22-dien-3β-ol) formed by gas chromatography–mass spectrometry with selected-ion monitoring. In carriers, the activity was significantly lower than in controls (22±2 vs 65±10 pmol/min per mg protein, p<0.0005), and no overlap was observed. The mean activity in carriers fibroblasts was more than 100 times higher than in patients cells (0.2 pmol/min per mg protein). The use of ergosterol avoids the many problems caused by the instability and lack of availability of radiolabelled 7-dehydrocholesterol. The present method makes it possible to discriminate SLOS carriers from both controls and patients using a commercially available substrate and common analytical equipment.

Effect of YM 9429, a potent teratogen, on cholesterol biosynthesis in cultured cells and rat liver microsomes

YM 9429 (cis-1-[4-(p-menthan-8-yloxy)phenyl]piperidine) is a hypolipidemic agent with a potent and specific teratogenicity, inducing cleft palate and skeletal variations in rats. Since cleft palate is generally observed in the Smith-Lemli-Opitz syndrome, a common syndrome of multiple congenital anomalies caused by reduced activity of 7-dehydrocholesterol delta 7-reductase (3 beta-hydroxysteroid delta 7-reductase), the final enzyme in the cholesterol biosynthetic pathway, YM 9429 was suspected of being an inhibitor of this enzyme. To prove this hypothesis, YM 9429 was added to cultured human skin fibroblasts and to cultured Morris hepatoma cells and incubated with [5-3H]mevalonolactone. After 24 h, radiolabeled 7-dehydrocholesterol accumulated in the cells, whereas the formation of radiolabeled cholesterol was markedly reduced. The results indicate that YM 9429 inhibits the conversion of 7-dehydrocholesterol to cholesterol catalyzed by the microsomal enzyme 7-dehydrocholesterol delta 7-reductase. In rat liver microsomes, the mode of inhibition was found to be noncompetitive, with a Ki of 40 microM. These results suggest that YM 9429 induced developmental abnormalities in rats by the same mechanism as the Smith-Lemli-Opitz syndrome. This compound might be useful for studying the pathogenesis of anomalies in animal models of the Smith-Lemli-Opitz syndrome.

Regulation of cholesterol biosynthetic pathway in patients with the Smith-Lemli-Opitz syndrome.

The Smith–Lemli–Opitz syndrome (SLOS) is a recessively inherited birth disorder caused by a defect in 7-dehydrocholesterol (3β-hydroxysteroid) Δ7-reductase, the final enzyme in cholesterol biosynthesis. To investigate in vivo regulation of the cholesterol biosynthetic pathway in SLOS, we measured hepatic microsomal sterol concentrations and activities of several key enzymes in the pathway, including HMG-CoA synthase, HMG-CoA reductase, squalene synthase and 7-dehydrocholesterol Δ7-reductase in liver specimens from a patient with SLOS and 11 controls. Hepatic microsomal 7-dehydrocholesterol Δ7-reductase activity in the patient was less than 1% of the control mean, and decreased cholesterol concentration and markedly increased 7- and 8-dehydrocholesterol concentrations were observed in the patients microsomes. HMG-CoA synthase and squalene synthase activities in the patient were upregulated to 149% and 532%, respectively, while the activity of HMG-CoA reductase, the rate-limiting enzyme in the pathway, was reduced to 39% of the control mean. Downregulation of HMG-CoA reductase activity in SLOS was supported by measuring plasma levels of mevalonic acid, the immediate product of HMG-CoA reductase. The levels in SLOS patients (n=9) were significantly low compared with age-matched controls (n=8) (12±2 vs 28±6 nmol/L, p<0.05). These results suggest that in most SLOS patients in vivo HMG-CoA reductase is not stimulated in spite of blocked cholesterol biosynthetic pathway and reduced plasma and hepatic cholesterol concentrations.