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PubMed2001

Fate of intravenously administered squalene and plant sterols in human subjects.

We have studied metabolism of plant sterols and squalene administered intravenously in the form of lipid emulsion mimicking chylomicrons (CM). The CM-like lipid emulsion was prepared by dissolving squalene in commercially available Intralipid. The emulsion was given as an intravenous bolus injection of 30 ml containing 6.3 mg of cholesterol, 1.9 mg of campesterol, 5.7 mg of sitosterol, 1.6 mg of stigmasterol, 18.1 mg of squalene, and 6 g of triglycerides in six healthy volunteers. Blood samples were drawn from the opposite arm before and serially 2.5 -180 min after the injections. The decay of CM squalene, plant sterols, and triglycerides was monoexponential. The half-life of CM squalene was 74 +/- 8 min, that of campesterol was 37 +/- 5 min (P < 0.01 from squalene), and those of sitosterol, stigmasterol, and triglycerides were 17 +/- 2, 15 +/- 1, and 17 +/- 2 min, respectively (P < 0.01 from squalene and campesterol). The CM squalene concentration still exceeded the baseline level 180 min after injection (P = 0.02), whereas plant sterols and triglycerides returned to the baseline level between 45 and 120 min after injection. The half-lives of squalene and campesterol were positively correlated with their fasting CM concentrations. In addition, VLDL squalene, campesterol, and triglyceride concentrations, VLDL, LDL, and HDL sitosterol concentrations, as well as VLDL and LDL stigmasterol concentrations were increased significantly. Cholesterol concentrations increased in VLDL (P < 0.05), but were unchanged in CM after injection. These data suggest that squalene clearance occurs more slowly than that of plant sterols and triglycerides from CM, and that squalene is more tightly associated with triglyceride-rich lipoproteins than are plant sterols in injected CM-like emulsions.

Adult↗
PubMed1990

Metabolic variables of cholesterol during squalene feeding in humans: comparison with cholestyramine treatment.

Squalene, a key intermediate of cholesterol synthesis, is present especially in olive oil. Regulation of cholesterol metabolism by dietary squalene in man is unknown, even though olive oil users in Mediterranean areas have low serum cholesterol levels. We have investigated absorption and serum levels of squalene and cholesterol and cholesterol synthesis with the sterol balance technique and serum levels of cholesterol precursors in humans during squalene feeding (900 mg/d for 7-30 days). The results were compared with those during cholestyramine treatment. Fecal analysis suggested that about 60% of dietary squalene was absorbed. Serum squalene levels were increased 17 times, but serum triglyceride and cholesterol contents were unchanged. The squalene feeding significantly (P less than 0.05) increased serum levels of free (1.7-2.3 times) and esterified (1.9-2.4 times) methyl sterol contents, while elevations of free and esterified delta 8-cholesterol and lathosterol levels were inconsistent. Cholestyramine treatment modestly augmented free methyl sterol levels (1.3-1.7 times), less consistently than those of esterified ones, while, in contrast to the squalene feeding, serum contents of free and esterified delta 8-cholesterol and lathosterol were dramatically increased (3.3-8 times). Neither of the treatments significantly affected serum plant sterol and cholestanol levels. The squalene feeding had no consistent effect on absorption efficiency of cholesterol, but significantly increased (paired t-test, P less than 0.05) the fecal excretions of cholesterol and its nonpolar derivatives coprostanol, epicoprostanol, and coprostanone (655 +/- 83 SE to 856 +/- 146 mg/d) and bile acids (212 +/- 24 to 255 +/- 24 mg/d), indicating an increase of cholesterol synthesis by about 50%. We suggest that a substantial amount of dietary squalene is absorbed and converted to cholesterol in humans, but this squalene-induced increase in synthesis is not associated with consistent increases of serum cholesterol levels. The clearly increased serum contents of esterified methyl sterols may reflect stimulated tissue acyl CoA: cholesterol acyltransferase (ACAT, EC 2.3.1.26) activity during squalene feeding as these sterols are not esterified in serum.

Cholestanol↗
PubMed1983

Absorption and metabolic fate of dietary 3H-squalene in the rat.

The absorption and metabolic fate of dietary squalene was investigated on the rat by administering a single oral dose of 3H-squalene and 14C-cholesterol. Experiments on rats with cannulated thoracic duct revealed that 3H-squalene was, like 14C-cholesterol, absorbed through the lymphatic vessels and that ca. 20% of absorbed 3H-squalene was cyclized to sterols during the transit through the intestinal wall. Feces contained 3H-sterols, indicating that newly synthesized mucosal sterols had been secreted into the gut lumen. In intact animals, 3H-squalene appeared in the circulation more rapidly than 14C-cholesterol and did not persist to any significant extent in the squalene-rich adipose and muscle tissues. The increase in dietary squalene load (8-48 mg) decreased the absorption percentage of 3H-squalene (45-26%) but did not affect the absorption of 14C-cholesterol (47%). Determination of fecal steroids revealed that during the first days absorbed 3H-squalene was eliminated to a significantly higher extent than 14C-cholesterol as fecal bile acids (34% vs 11%). The experiments indicate that the rat intestine has a marked capacity for absorbing dietary squalene and that the absorbed squalene is preferentially converted to bile acids in the liver.

Animals↗
PubMed2003

In vitro cytoprotective activity of squalene on a bone marrow versus neuroblastoma model of cisplatin-induced toxicity. implications in cancer chemotherapy.

The development of a non-toxic selective cytoprotective agent that preferentially protects normal tissues from chemotherapy toxicity, without protecting malignant tissues, is a major challenge in cancer chemotherapy research. The available cytoprotective agents are either toxic or lack selective cytoprotective activity. Here, we report the in vitro selective cytoprotective activity of squalene, an isoprenoid molecule with antioxidant properties. Normal human bone marrow (BM) derived colony-forming unit (CFU) growth was increased by squalene in a dose-dependent manner. Squalene (12.5-25 microM) treatment significantly protected the CFUs from cisplatin-induced toxicity; the protective effect was equivalent to reduced glutathione (GSH), a known cytoprotective agent. Squalene also increased the long-term survival of cisplatin-treated 4-week-old CFUs. Cisplatin-induced apoptosis of CFUs as measured by the TUNEL assay was reduced by squalene. To examine the squalene-induced protection of tumours, several neuroblastoma cell lines, including five MYCN-amplified cell lines, were grown in monolayers, as well as in anchorage-independent cultures, in the presence of squalene and cisplatin. Squalene did not protect the neuroblastoma (NBL) cell lines from cisplatin-induced toxicity. In addition, squalene did not protect the NBL cells from carboplatin, cyclophosphamide, etoposide and doxorubicin-induced toxicity. In conclusion, our results suggest that squalene has a selective in vitro cytoprotective effect on BM-derived haematopoietic stem cells that is equipotent to GSH.

Antineoplastic Agents↗
PubMed2005

The protective role of squalene in alcohol damage in the chick embryo retina.

The developing CNS, and in particular the visual system, is very sensitive to the effects of alcohol. Alcohol causes lipid peroxidation. Squalene, the major olive oil hydrocarbon, is a quencher of singlet oxygen and prevents the corresponding lipid peroxidation. We presumed that squalene can protect against the alcohol-induced damage already observed during the development of the chick retina. Alcohol+squalene was administered directly into the yolk sac of the egg of White Leghorn chicks at day 6 of incubation. The lipid composition of the retina was analyzed in embryos at E7, E11, E15 and E18. The proportions of phospholipids, free and esterified cholesterol, diacylglycerides and free fatty acids were estimated using the Iatroscan TLC/FID procedure. Gas chromatography and mass spectrometry were used to determine the fatty acid composition. The morphological study was carried out at E11 using semithin sections, and by means of immunohistochemical techniques at E19. Comparing the results obtained in control embryos, the administration of alcohol+squalene reduces the effects of alcohol on the total lipid composition of the retina during development. The effects were, in fact, of less magnitude than in embryos treated only with alcohol. The major phospholipid species of alcohol+squalene-treated embryos exhibited total recuperation at E15. As far as fatty acids are concerned, no significant changes were observed with regard to control embryos during development. From a morphological point of view, the retinas of alcohol+squalene-treated embryos show at E11 fewer cellular alterations than the retinas of alcohol-treated embryos. In this respect, the retinas of alcohol+squalene-treated embryos exhibited: a columnar cell arrangement similar to that observed in control retinas; few pycnotic cells and very few alterations in ganglion cell layers and in the optic nerve fibers layer. At E19 the recuperation of the expression of myelin oligodendrocyte specific protein (MOSP) in alcohol+squalene-treated embryos was recorded. Since squalene reduces the deleterious effects caused by alcohol on the lipid composition and the structure of the retina, squalene could act as a naturally occurring agent for the prevention of damage caused by abusive alcohol ingestion during pregnancy.

Animals↗
PubMed2002

Extraction and purification of squalene from amaranthus grain.

Grain amaranth has been suggested as an alternative to marine animals as a natural source of squalene. Oil contents, squalene contents, and fatty acid profiles were determined in 11 genotypes of four grain amaranth species. Although the oil contents of grain amaranth were low (from 5.1% in Amaranthus tricolor to 7.7% in Amaranthus cruentus) as compared to other oil-containing grains, high concentrations of squalene were found in total lipids, ranging from 3.6% in Amaranthus hypochondriacus to 6.1% in A. tricolor. The major fatty acids in Amaranthus oil consisted of palmitic acid (19.1-23.4%), oleic acid (18.7-38.9%), and linoleic acid (36.7-55.9%). A high degree of unsaturation was observed in Amaranthus oils, with S/U ratios of 0.26-0.32. A method to isolate and purify the squalene from Amaranthus oil was developed. After the saponification of K112, the squalene content increased from 4.2% in the crude oil to 43.3% in the unsaponifiables by the removal of the saponifiables. The unsaponifiables were fractionated by silica gel column chromatography to get highly purified squalene. The squalene purity in certain fractions was as high as 98%. Combining the fractions rich in squalene gave a 94% squalene concentrate, with a yield of 90%. The structure of squalene in the purified sample was confirmed by comparison of its ultraviolet spectrum with a standard and from its nuclear magnetic resonance spectra.

Amaranthus↗
PubMed2003

Ultraviolet a induces generation of squalene monohydroperoxide isomers in human sebum and skin surface lipids in vitro and in vivo.

At the outermost surface of human skin, skin surface lipids are first-line targets of solar ultraviolet radiation. Therefore, we hypothesized that ultraviolet A and ultraviolet B irradiation induce photo-oxidation of skin surface lipids. To test this, sebum samples were collected from facial skin of 17 healthy volunteers, weighed, and immediately irradiated with either ultraviolet B or ultraviolet A. Squalene, the major sebum lipid, as well as photo-oxidation products were identified in sebum lipid extracts by high-performance liquid chromatography analysis. Upon ultraviolet A exposures squalene was depleted in a concentration-dependent manner, whereas an unidentified sebum lipid photo-oxidation product was detected. Using high-performance thin layer chromatography, high-performance liquid chromatography, atmospheric pressure chemical ionization mass spectrometry, and nuclear magnetic resonance, unidentified sebum lipid photo-oxidation product was identified as a mixture of squalene monohydroperoxide isomers. Squalene monohydroperoxide isomers purified from sebum were identical with squalene monohydroperoxide isomers synthesized by preparative photo-oxidation of squalene. Squalene monohydroperoxide isomers were formed even after small suberythematogenic doses of ultraviolet A (5 J per cm2). Whereas physiologic baseline levels of squalene monohydroperoxide isomers in human skin were only slightly above detection limits, squalene monohydroperoxide isomer levels were strongly increased by suberythematogenic doses of ultraviolet A both in vitro and in vivo. High-performance liquid chromatography results could be complemented by a straightforward thin layer chromatography method for rapid screening of lipid peroxide formation in human sebum/skin surface lipids. In conclusion, specific squalene monohydroperoxide isomers were identified as highly ultraviolet A sensitive skin surface lipid breakdown products that may serve as a marker for photo-oxidative stress in vitro and in vivo.

Adult↗
PubMed1991

Tellurium blocks cholesterol synthesis by inhibiting squalene metabolism: preferential vulnerability to this metabolic block leads to peripheral nervous system demyelination.

Inclusion of 1.1% elemental tellurium in the diet of postweanling rats produces a peripheral neuropathy due to a highly synchronous primary demyelination of sciatic nerve; this demyelination is followed closely by remyelination. Sciatic nerves from animals fed tellurium for various times were removed and incubated ex vivo for 1 h with [14C]acetate, and radioactivity incorporated into individual lipid classes was determined. In nerves from rats exposed to tellurium, there was a profound and selective block in the conversion of radioactive acetate to cholesterol. Another radioactive precursor, [3H]water, gave similar results. We suggest that tellurium feeding inhibits squalene epoxidase activity and that the consequent lack of cholesterol destabilizes myelin, thereby causing destruction of the larger internodes. Ex vivo incubation experiments were also carried out with liver slices. As with nerve, tellurium feeding caused accumulation in squalene of label from radioactive acetate, whereas labeling of cholesterol was greatly inhibited. Unexpectedly, however, incorporation of label from [3H]water into both squalene and cholesterol was increased. Relevant is the demonstration that liver was the primary site of bulk accumulation of squalene, which accounted for 10% of liver dry weight at 5 days. Thus, accumulation of squalene (and other mechanisms, possibly including up-regulation of cholesterol biosynthetic pathways) drives squalene epoxidase activity at normal levels in liver even in the presence of inhibitors of this enzyme. This is reflected by continuing incorporation of [3H]water into cholesterol; incorporation of this precursor takes place at many of the postsqualene biosynthetic steps for sterol formation. [14C]Acetate entering the sterol pathway before squalene in liver is greatly diluted in specific activity when it reaches the large squalene pool, and thus increased squalene epoxidase activity does not transfer significant 14C label to sterols. In contrast to the situation with liver, synthesis of sterols is markedly depressed in sciatic nerve, and squalene does not accumulate to high levels.

Acetates↗
PubMed1993

Enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae.

Squalene epoxidase is a microsomal membrane-associated enzyme that acts as an important regulator in the sterol biosynthetic pathway. In this study, the enzymatic properties of squalene epoxidase from Saccharomyces cerevisiae were examined. Unlike Candida squalene epoxidase, S. cerevisiae squalene epoxidase required NADPH for enzyme reaction. However, S. cerevisiae enzyme reaction did not require FAD or autologous S105 fraction. Unlike rat squalene epoxidase, the activity of S. cerevisiae was reduced by Triton X-100, a nonionic detergent. Terbinafine, an inhibitor of fungal squalene epoxidase, inhibited the enzyme in a non-competitive manner, while NB-598, an inhibitor of mammalian squalene epoxidase, barely inhibited it in a partially non-competitive manner. Thus, the properties of squalene epoxidase from S. cerevisiae were different from those of squalene epoxidase from rats and Candida, which were previously known. We propose that a species difference of squalene epoxidase exists not only between animals and fungi but between Candida and Saccharomyces.

Animals↗
PubMed1980

Supernatant protein factor facilitates intermembrane transfer of squalene.

Squalene epoxidation of microsome-associated squalene is stimulated by a soluble protein termed "supernatant protein factor" (SPF) (Saat, Y. A., and Bloch, K. E. (1976)J. Biol. Chem. 251, 5155-5160). In the absence of SPF, the initial rate for microsome-bound squalene epoxidation is rapid for 5 to 10 min but falls off sharply thereafter. SPF does not affect the rapid initial epoxidation rate of reaction but maintains it for longer periods. This SPF effect on enzyme kinetics indicates that SPF facilitates the otherwise rate-limiting access of squalene to the epoxidse site. Trypsin treatment of microsomes totally inactivates squalene epoxidase. When such trypsin-treated squalene-containing microsomes are incubated with normal, squalene-free, enzymatically active microsomes, formation of squalene epoxide is not observed. However, if SPF is included in this system, conversion of squalene to 2,3-oxidosqualene occurs rapidly. Lowering the temperature from 37 degrees to 22 degrees C abolishes the SPF effect in assay systems containing either normal or trypsin-treated plus normal microsomes. These findings show that SPF promotes the transfer of squalene from one microsome population to another, i.e. intermembrane transfer of substrate.

Animals↗
PubMed1993

Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence.

In the present study we investigated the subcellular localization of squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21). Squalene synthase catalyzes the formation of squalene from trans-farnesyl diphosphate in two distinct steps and is the first committed enzyme for the biosynthesis of cholesterol. Recently, a truncated form of the enzyme from rat hepatocytes was purified, and monospecific antibodies for squalene synthase were produced. This enabled the subcellular localization of squalene synthase by three different methods: (i) analytical subcellular fractionation and measurements of enzyme activities; (ii) immunodeterminations of squalene synthase in the isolated subcellular fractions with a monospecific antibody; and (iii) immunoelectron microscopy. All three methods gave consistent results. The data clearly illustrate that squalene synthase enzymatic activity and squalene synthase are exclusively localized in the endoplasmic reticulum. In rat hepatic peroxisomes we were not able to detect any squalene synthase. In addition, we also demonstrated that squalene synthase in the microsomal fraction is dramatically regulated by a number of hypolipidemic drugs and dietary treatments.

Animals↗
PubMed1979

Squalene synthetase.

In the first part of the review the background to the discovery of the asymmetric synthesis of squalene from two molecules of farnesyl pyrophosphate and NADPH is described, then the stereochemistry of the overall reaction is summarized. The complexity of the biosynthesis of squalene by microsomal squalene synthetase demanded the existence of some intermediate(s) between farnesyl pyrophosphate and squalene. This demand was satisfied by the discovery of presqualene pyrophosphate, an optically active C30 substituted cyclopropylcarbinyl pyrophosphate, the absolute configuration of which at all three asymmetric centers of the cyclopropane ring was deduced to be R. Possible mechanisms for the biosynthesis of presqualene pyrophosphate and its reductive transformation into squalene are presented. In the second part of the review the nature of the enzyme is discussed. The question whether presqualene pyrophosphate is an obligate intermediate in the biosynthesis of squalene is examined, with the firm conclusion that it is. It is as yet uncertain whether the two half reactions of squalene synthesis, i.e. (i) 2 x farnesyl pyrophosphate leads to presqualene pyrophosphate; (ii) presqualene pyrophosphate + NADPH (NADH) leads to squalene, are catalyzed by one or two enzymes or by a large complex with two catalytic sites. Evidence is cited for the existence on the enzyme of two distinct binding sites with different affinities for the two farnesyl pyrophosphate molecules. The types of enzyme preparations available at present are described and types of experiments carried out with these are critically examined. The implications of the properties of a low molecular weight squalene synthetase solubilized with deoxycholate from microsomal membranes is discussed and a model for the enzyme in an organized membrane structure is presented.

Animals↗
PubMed1993

Squalene promotes the formation of non-bilayer structures in phospholipid model membranes.

A study of the lipid polymorphism of aqueous dispersions of stearoyloleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylchloline (95:5, mol/mol) in the presence of the hydrophobic molecule squalene, an intermediate in the biosynthesis of sterols, has been performed. With increasing concentration of squalene the main transition temperature was decreased from 29.8 degrees C for the pure phospholipid system to 28.1 degrees C for samples containing 5 mol% squalene without considerable changes in the phase transition enthalpy as detected by high precision differential scanning calorimetry. The structure of the phospholipid aggregates was determined by small- and wide-angle X-ray diffraction experiments showing only a minor increase of the lamellar repeat distance of the liquid-crystalline phase for the squalene containing samples. By far more pronounced was the effect of squalene on the lamellar-to-inverse-hexagonal phase transition, which was shifted from 64 degrees C to about 36 degrees C in the presence of 6 mol% squalene, thereby overlapping with the main transition. X-ray data showed that the size of the tubes of the inverse hexagonal phase are increasing linearly up to 6 mol% squalene. Experiments performed in the presence of 10 mol% squalene did not further change the phase behaviour, indicating the limiting partition of this hydrophobic molecule into the membrane. The high efficiency of squalene to promote the formation of the inverse hexagonal phase is discussed along the lines of argument based on the model of Kirk et al. (Kirk, G.L., Gruner, S.M. and Stein, D.E. (1984) Biochemistry 23, 1093-1102).

Calorimetry, Differential Scanning↗
PubMed1981

Protein-facilitated intermembrane transfer of squalene. Demonstration by density gradient centrifugation.

Squalene-enriched, trypsinized microsomes display no squalene epoxidase activity either as such or when combined with normal microsomes. On addition of microgram quantities of supernatant protein factor to the combined system, squalene epoxidation commences at once and continues at a rapid rate (Friedlander, E. J., Caras, I. W., Lin, L. F., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). When mixtures of trypsin-treated, [3H]squalene-containing microsomes and normal microsomes are subjected to isopycnic density gradient centrifugation, the two microsomal populations separate readily. Essentially all of the radioactive squalene remains associated with the lighter (trypsinized) fraction of microsomes. However, if the mixture of microsomes is initially incubated with supernatant protein factor and then centrifuged, a large fraction of labeled squalene sediments with the denser, normal microsomes. Thus, supernatant protein factor mediates the transfer of squalene from one microsome population to another. This conclusion had previously been reached on the basis of less direct experiments (Friedlander, E. J., Caras, I. W., Lin, L. F., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045). Evidence is presented that the process of supernatant protein factor-mediated squalene transfer does not involve membrane fusion and proceeds also in the reverse direction.

Animals↗
PubMed1993

Radioprotection of mice by dietary squalene.

Male C3H mice were fed a diet containing 2% squalene for 14 d prior to and 30 d subsequent to exposure to 6, 7 or 8 Gy of whole body gamma-irradiation (Cesium-137). After 14 d on squalene-supplemented diet, plasma and jejunal tissue squalene levels were 2X and 15X that of controls. Seven days after irradiation, total white cell counts and total lymphocyte counts were substantially depressed in a radiation dose-dependent manner. Although counts in the squalene group were consistently (18-119%) higher than those in the corresponding dietary control group, the differences between dietary groups at any single dose were not significant. Nuclear area of villus cells in the jejunum of both dietary groups was significantly reduced (20%) by day 11 post-irradiation but the nuclear area in squalene-fed mice was significantly greater (15%) than in controls, before and after irradiation. There were no differences in body weight as a function of either diet or radiation dose prior to the first observations of animal lethality. Animal survival was decreased from 100 to 0% at 30 d post-irradiation by radiation doses of 6-8 Gy, with the greatest difference between dietary groups being observed at 7 Gy (median survival times of 12 and 16 d for control and squalene groups, respectively). Overall, survival of squalene-fed mice was significantly prolonged compared with control-fed mice (P = 0.0054 by censored multiple regression analysis). It is concluded that squalene conferred some cellular and systemic radioprotection to mice receiving these lethal whole-body radiation doses.

Animals↗
PubMed1995

Isoprenoid lipid metabolism in the retina: dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes.

Frogs were injected intravitreally with [3H]acetate, and the formation of [3H]-labeled squalene and cholesterol in the retina and their incorporation into rod outer segment (ROS) membranes were evaluated biochemically over a 60-day time course. ROS [3H]squalene specific activity was maximal by 1-3 days, then declined with a half-time of approximately 20-30 days. In contrast, the specific activity of ROS [3H]cholesterol initially increased to a level substantially less than that of [3H]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS cholesterol. When [3H]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% of those recovered from the ipsilateral retina; hence, systemic contributions to de novo synthesis were obviated. Long-term (> or = 8 hr) in vitro incubations of isolated retinas with [3H]acetate resulted in incorporation of [3H]-labeled sterols and squalene into ROS, at levels comparable to those observed in ROS from companion incubated eyecup preparations and from retinas 8 hr after intravitreal injection of [3H]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with respect to isoprenoid lipid metabolism, and suggest that de novo synthesis within the neural retina is responsible for generating most, if not all, of the [3H]squalene and [3H]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy poisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited marked suppression of incorporation of newly synthesized protein into the ROS, while [3H]squalene incorporation was only minimally reduced, compared with frogs maintained at 22 degrees C. These results are consistent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane proteins.

Animals↗
PubMed1975

Measurement of cholesterol synthesis in man by isotope kinetics of squalene.

A method for measuring the rate of total daily cholesterol synthesis in man has been developed through isotope kinetic studies of squalene biosynthesis after intravenous administration of [14C]mevalonic acid. Plasma squalene becomes rapidly labeled, reaching maximal specific activity approximately 100 min after mevalonate administration and then decays exponentially to reach undetectable levels in 12 hr. The rate of daily squalene synthesis equals the percent dose of mevalonate converted to cholesterol divided by the area under the specific activity curve of squalene; the fraction of the dose of mevalonate converted to cholesterol is calculated by the simultaneous injection of [3H]- and [14C] cholesterol in plasma. The premise that squalene and cholesterol synthetic rates are equivalent was tested. In seven patients it was found that the mean daily cholesterol synthesis rates estimated simultaneously by sterol balance and by sqyalene kinetic methods agreed within 8%. In addition, fractional conversions of mevalonic acid to cholesterol were highly correlated with cholesterol synthesis rates. Maximum estimates of the pool sizes and half-lives of metabolically "active" squalene also were obtained. This measurement of daily cholesterol synthesis by squalene kinetics minimizes patient inconvenience, is suitable for outpatient studies, and yields results in 4 weeks or less. Because of the rapidity of the rate of squalene synthesis, the results obtained reflect cholesterol synthesis over a period of less than 10 hr and are therefore uniquely applicable to unsteady state situations.

Adult↗
PubMed1998

Dual localization of squalene epoxidase, Erg1p, in yeast reflects a relationship between the endoplasmic reticulum and lipid particles.

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 x g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Delta24-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Amino Acid Sequence↗