M. Galli Kienle

The effect of cholestyramine on liver HMG-CoA reductase and cholesterol 7α-hydroxylase in various laboratory animals

The activity of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was assayed in the liver of rats, rabbits, hamsters and guinea pigs at the minimum of the day cycle and after one night fasting. The amount of HMG-CoA reductase, as determined after its complete dephosphorylation in vitro was of the same order of magnitude in the tested species. The dephosphorylated active form of the enzyme was detectable only in the rat. Cholesterol 7 alpha-hydroxylase activity was also much higher in the rat. Cholestyramine treatment stimulated the activity of both enzymes. In particular, the ratio between active and inactive HMG-CoA reductase in rabbits, hamsters and guinea pigs became of the same order of magnitude of that found in rats.

Hydrogen Exchange and Double Bond Formation in Cholesterol Biosynthesis

The conversion of lanosterol║to cholesterol requires a considerable number of intermediary steps involving loss or uptake of hydrogen atoms and formation and migration of nuclear double bonds. Detailed discussions on the intermediary steps in cholesterol biosynthesis are reported in several reviews (Olson 1965; Frantz & Schroepfer 1967; Goad 1970). In the present report some mechanisms in the formation of cholesterol and its sterol precursors from lanosterol are discussed. The relation between in vitro and in vivo pathways of cholesterol biosynthesis and the composition and metabolism of sterols in biological tissues is underlined.

Determination of plasma [6,6-2H2]glucose enrichment by a simple and accurate gas chromatographic-mass spectrometric method

An improved method for the evaluation of glucose turnover rate in humans, using a prime-continuous infusion of [6,6-2H2]glucose, was developed. Deproteinization of plasma and conversion of glucose into the aldononitrile pentaacetate derivative are the only sample manipulations required prior to the gas chromatographic-mass spectrometric analysis. In six normal adults (prime = 5 mg kg-1; continuous infusion = 0.05 mg kg-1 min-1) the hepatic glucose output calculated at steady state by the procedure described here was 2.1 +/- 0.2 mg kg-1 min-1, the isotopic enrichment being determined with a coefficient of variation of ca. 2%. In six additional subjects, with half of the above-mentioned doses of labelled glucose, the mean hepatic glucose output was exactly the same (3.2% coefficient of variation for the isotopic enrichment measurement). The method described allows the hepatic glucose output to be precisely determined with savings both of time and of labelled glucose.

Inhibition of cholesterol synthesis and hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in rats by simvastatin and pravastatin

In this communication we attempt to provide one possible explanation for the observed differences regarding kinetics and distribution between simvastatin and pravastatin. Rats treated with simvastatin or pravastatin exhibited a reduction in the incorporation of [2-14C]acetate into liver cholesterol and displayed lower plasma mevalonate levels as compared to control animals. Moreover, both the total and dephosphorylated 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.34) activities, particularly 1 h after treatment, were greatly reduced in liver microsomes obtained from simvastatin-treated as compared to control rats. During the same time frame, these parameters were actually elevated with pravastatin treatment. It is known that HMG-CoA reductase synthesis and activity increase following their competitive inhibition. Our results suggest that pravastatin, at 1 h following treatment, was no longer bound to the enzyme; however, it had entered the liver because its inhibitory effect on cholesterol synthesis was manifest at early times after administration. These data provide a plausible rationale for the earlier observation that activity of simvastatin persists longer in plasma than does that of pravastatin.

Increased appearance rate of 27-hydroxycholesterol in vivo in hypercholesterolemia: A possible compensatory mechanism

BACKGROUND AND AIMS The first step in the alternative pathway of bile acid biosynthesis is the 27-hydroxylation of cholesterol, which takes place both in liver and extrahepatic tissues. This pathway is believed to play a role in peripheral cholesterol degradation. Aim of this study was to investigate the impact of hyperlipidemia on 27-hydroxycholesterol appearance rate, and to assess the effects induced by treatment with statins. METHODS AND RESULTS Seven patients with familial hypercholesterolemia and eight patients with familial combined hyperlipidemia underwent determination of 27-hydroxylation rates in vivo by i.v. infusion of deuterated 27-hydroxycholesterol. Isotope enrichment was assayed by gas chromatography-mass spectrometry, allowing to calculate 27-hydroxycholesterol appearance rates. Six normocholesterolemic subjects were regarded as controls. In some hypercholesterolemic patients the infusions were repeated during treatment with atorvastatin or rosuvastatin. Hydroxylation rates were higher in hypercholesterolemic patients (8.7 ± 2.5 mg/h; controls, 3.4 ± 2.0 mg/h; combined hyperlipidemia, 4.4 ± 1.6 mg/h; mean ± SD, P < 0.01 vs both). After statin treatment, both plasma cholesterol levels and hydroxylation rates dropped by nearly 50%. No difference was detectable between the two statins. A linear correlation was shown between plasma cholesterol and 27-hydroxylation rates. CONCLUSION Hypercholesterolemia associates with increased 27-hydroxycholesterol appearance rates, which decrease during hypocholesterolemic treatment. The correlation with cholesterol levels supports the view that 27-hydroxylation may act as a compensatory mechanism in a condition of larger plasma cholesterol pool. A regulatory role for hepatic and extrahepatic nuclear receptors seems reasonable. These data prompt novel pharmacological approaches for the management of hypercholesterolemia and the prevention of atherosclerosis.

Effects of Pantethine on Cholesterol Synthesis from Mevalonate in Isolated Rat Hepatocytes

Results presented here show that when isolated rat hepatocytes are incubated with increasing concentrations of [2-14C]mevalonolactone, incorporation of the substrate into cholesterol is progressively reduced. Correspondingly, an increase of the incorporation of the substrate into precursors of cholesterol (methyl sterols and squalene) occurs. These effects and the observed inhibition of HMGCoA reductase at high mevalonolactone concentration (0.5 mM) are in agreement with those shown by others in cultured hepatocytes. Since pantethine was reported to affect cholesterol biosynthesis from mevalonate in cultured fibroblasts, effects of its addition to hepatocyte incubations at low and high mevalonolactone concentration were studied. Neither the amount of radioactivity incorporated into cholesterol and in its sterol precursors nor sterol levels were modified by pantethine when a mevalonolactone concentration (0.01 mM) that did not alter the levels of intermediates of cholesterol synthesis was used. Pantethine was shown instead to potentiate the decrease of mevalonate incorporation into cholesterol induced by high concentrations of mevalonolactone (0.5 mM). Decrease of 3-hydroxy-3-methylglutaryl CoA reductase activity induced by 1 mM pantethine was twice that caused by mevalonolactone alone. These results may explain the fact that both in laboratory animals and in humans pantethine administration is effective in reducing cholesterol plasma levels in hyperlipidemic conditions.

The stereochemistry of hydrogen elimination in the biological conversion of 5α-cholest-8-en-3β-ol to 5α-cholest-7-en-3β-ol

Abstract The stereochemistry of 5α-cholest-8-en-3β-ol to 5α-cholest-7-en-3β-ol isomerization was studied in rat liver homogenates incubated in the presence of either 3(±)-2R-[2- 3 H 1 ]- or 3(±)-2S-[2- 3 H 1 ]-mevalonic acid lactone. The isolated radioactive 5α-cholest-7-en-3β-ol and cholesta-5,7-dien-3β-ol acetates were transformed into the mixtures of epimeric cis-5α-cholestane-3β,7,8-triol-3β-acetates and then into the mixtures of epimeric 5α-cholestane-3β,8-diol-7-one 3β-acetates. Radioactivity contents showed that during the biological isomerization the 7α-hydrogen of 5α-cholest-8-en-3β-ol is completely eliminated whereas the 7β-hydrogen is retained.

A rapid, sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol.

Abstract A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at m e 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 ± 49 and 979 ± 15 nmol· g tissue −1, respectively. Both total and XDH activities in ischémie kidneys (30 ± 15 and 19 ± 1 nmol·min−1 ‐g tissue−1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type‐O activity of xanthine oxidase.

Effects of dietary soy protein on liver catabolism and plasma transport of cholesterol in hypercholesterolemic rats.

Abstract Sprague-Dawley rats were fed hypercholesterolemic regimens with either soy protein (S) or casein (HC) as the protein source. Lipid, lipoprotein composition as well as lecithin cholesterol acyltransferase activity (LCAT) were measured in plasma. Liver lipid levels and enzyme activities, cholesterol 7α-hydroxylase and acylCoA cholesterol acyltransferase (ACAT), were also determined. Plasma cholesterol levels were significantly reduced with the S as compared with the HC diet, but LCAT activity was not modified. Cholesterol 7α-hydroxylase, which increases in animals fed hypercholesterolemic diets as compared to controls, was unaffected when S was substituted for casein. In contrast, higher cholesterol ester levels and an elevated ACAT activity were observed in the liver of rats on the soybean diet. Substitution of casein with soy protein reduced cholesterol levels in all lipoprotein fractions but no changes were observed in apoproteins.