Capillary GC quantification of cholesterol oxidation products in plasma lipoproteins of fasted humans

Cholesterol oxidation products have been hypothesized to be important factors in atherosclerosis, a process which can culminate in myocardial infarction. The relative importance of exogenous or in vivo sources of cholesterol oxidation products has not been determined. However, methodology used for cholesterol oxidation products analysis of foods is applicable to the determination of cholesterol oxidation products in human plasma lipoproteins. Such methodology, outlined in this report, permits numerous critical experiments to be conducted on the possible role of cholesterol oxidation products in coronary heart disease.     

The oxygenation of cholesterol esters by the reticulocyte lipoxygenase

The arachidonate 15-lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV-spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.     

Rat granulosa cell apolipoprotein E secretion. Regulation by cell cholesterol

We have demonstrated previously that cultured rat ovarian granulosa cells synthesize and secrete apoE, and this production of apoE is increased by agents that stimulate protein kinase A (cyclic AMP-dependent enzyme) (for example, cholera toxin) and protein kinase C (Ca2+/phospholipid-dependent enzyme) (for example, 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester). In the studies presented in this report, we have examined the effect of changes in cell cholesterol synthesis on the production of apoE by rat ovarian granulosa cells. Mevinolin, an inhibitor of hydroxymethylglutaryl (HMG)-CoA reductase (the rate-limiting enzyme in cholesterol synthesis), and 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of squalene cyclization, both attenuate the cholera toxin or 12-O-tetradecanoylphorbol-13-acetate stimulation of granulosa cell apoE secretion and apoE mRNA content in a dose-responsive manner. The inhibitory effect of mevinolin is reversed by the concomitant administration of mevalolactone, which provides the cells with the product of the reaction catalyzed by HMG-CoA reductase. Steroidogenesis per se has no effect on apoE production. Aminoglutethimide, which blocks the rate-limiting step in steroidogenesis, has no effect on apoE or apoE mRNA. The data indicate that products of HMG-CoA reductase (isoprenes, cholesterol and/or cholesterol metabolites) are required along with stimulators of protein kinases A and C, to regulate ovarian granulosa cell apoE production.     

Adaptive response induced by lipid peroxidation products in cell cultures

The adaptive response induced by the lipid peroxidation products, such as phosphatidylcholine hydroperoxide, lysophosphatidylcholine (LysoPC), 15-deoxy-Delta(12,14)-prostaglandin J(2), 4-hydroxynonenal (4-HNE), hydroxyoctadecadienoic acid, 7-hydroxycholesterol, and cholesterol 5beta,6beta-epoxide, was investigated in this study. Although these products have been implicated in oxidative stress-related diseases, pretreatment with such compounds at sublethal concentrations significantly protected PC12 cells against subsequent oxidative stress induced by 6-hydroxydopamine. Moreover, 4-HNE and LysoPC also exhibited adaptive protection in human arterial endothelial cells. These findings suggest a general hormetic effect of such compounds in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.     

Genetic heterogeneity in Niemann-Pick C disease: a study using somatic cell hybridization and linkage analysis.

The primary molecular defect underlying Niemann-Pick C disease (NPC) is still unknown. A wide spectrum of clinical and biochemical phenotypes has previously been documented. Indication of genetic heterogeneity has recently been provided for one patient. In the present study, somatic cell hybridization experiments were carried out on skin fibroblast cultures from 32 unrelated NPC patients covering the range of known clinical and biochemical phenotypes. The criterion for complementation was the restoration of a normal intracellular fluorescent pattern in polykaryons stained with filipin to document cholesterol distribution. Crosses between the various cell lines revealed a major complementation group comprising 27 unrelated patients and a second minor group comprising 5 patients. Linkage analysis in one multiplex family belonging to the minor complementation group showed that the mutated gene does not map to the 18q11-12 region assigned to the major gene. Patients in the first group spanned the whole spectrum of clinical and cellular phenotypes. No consistent clinical or biochemical phenotypes was associated with the second complementation group. Three of the five group 2 patients, however, presented with a new rare phenotype associated with severe pulmonary involvement leading to death within the first year of life. No biochemical abnormality specific of either group could be demonstrated with regard to tissue lipid storage pattern, intralysosomal cholesterol storage, and regulation of cholesterol homeostasis. Mutations affecting at least two different genes have thus been shown to underlie NPC. The two gene products may function together or sequentially in a common metabolic pathway affecting intracellular cholesterol transport.     

Cholesterol removal capability of lactic acid bacteria and related cell membrane fatty acid modifications

Milk and dairy products play an important role in a healthy diet because of their high nutritional value, even if they represent a source of lipids and cholesterol. Nowadays, some commercially hypocholesterolemic products are available, which contain lactic acid bacteria (LAB). Therefore, the aims of this study were to test and compare the cholesterol removal abilities of different LAB species and to investigate the capacity of the cholesterol to change the cellular fatty acid composition of microorganisms. Fifty-eight strains of dairy LAB were studied for their ability to remove cholesterol during 24 h of growth. Two of them, L. plantarum 885 and L. acidophilus LA-5®, showed the higher reduction capability. For these strains, the cellular fatty acid composition was studied. They showed a different behaviour, which appeared related to the needs of the cells to maintain the characteristics of membrane fluidity, but was dependent upon their original fatty acid composition. Further studies are required to better characterise the LAB strains to be used to develop fermented dairy products with reduced cholesterol content or be able to induce hypocholesterolemic effects. It will also be interesting to investigate the possible modifications of the cell membrane caused by cholesterol and its possible involvement in cell metabolism.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant

Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3beta,7a-diol, and 5beta,6beta-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5beta,6beta-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5beta,6beta-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3beta,5alpha,6beta-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3beta,5alpha-diol. Both 5beta,6beta-epoxycholesterol and cholestan-6-oxo-3beta,5alpha-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells.     

Somatic cell genetics and the study of cholesterol metabolism

The regulation of cholesterol biosynthesis by extracellular cholesterol occurs both in whole animal tissue and in permanent somatic cell lines in culture. Permanent mammalian cells lines, under optimized growth conditions, are easily manipulated both biochemically and genetically. The Chinese hamster ovary cell line (CHO-K1) is the most widely used cell line for genetic studies. CHO-K1 is a pseudo-diploid mammalian cell exhibiting a short doubling time and a relatively high plating efficiency. Somatic cell mutants can be generated through mutagenesis and also by drug adaptation. Following mutagenesis, auxotrophs may be isolated either by selection or by screening. Most selection procedures for mutants of cholesterol metabolism must be done in serum depleted of cholesterol which requires the endogenous biosynthetic pathway to be intact. Mutants failing to produce cholesterol do not replicate their DNA and exhibit reduced concentrations of cholesterol in their membranes. BUdR and polyene antibiotics have both been used to select against the wild-type cells which incorporate these compounds and are killed, allowing the survival of the mutant cells. Both mevalonate and cholesterol auxotrophs have been isolated with the BUdR technique and have proven useful for elucidation of the early steps in cholesterol biosynthesis, particularly for the ratelimiting enzyme HMG-CoA reductase. Somatic cell fusion of a mutant and wild-type cell followed by chromosomal segregation, routinely used to map human genes, has also been used to map the human gene for HMG-CoA synthase. Such hybrids also provide valuable information on the dominance or recessivity of a specific lesion. DNA-mediated gene transfer into somatic cell mutants allows the selection of DNA sequences which complement the mutation, and is also useful for analysis of regions of regulatory significance. Mutants, resistant to the regulatory effects of oxygenated sterols, can be isolated following mutagenesis. Mutants of this type vary the lipid content of their membranes in response to cholesterol concentration in the medium. All such mutants tested exhibit a pleiotropic regulatory effect on more than one enzyme in the cholesterol biosynthetic pathway. Adaptation to drugs such as compactin and mevinolin, which inhibit HMG-CoA reductase, have been used to produce mutants which overexpress enzymes in the pathway. These amplified cells are useful sources of specific mRNAs for construction of cDNA libraries and gene isolation. Structure-function relationships of membrane sterols can be studied in cholesterol auxotrophs where changes in acyl-chain ordering can be manipulated by exogenous sterols in the medium.     

Decrease in the degree of lipoid infiltration of rabbit tissues during feeding with cholesterol freed from autoxidation products

Lipid accumulation in the rabbit liver and myocardium has been investigated by histochemical and biochemical methods. The rabbits received cholesterol that contained 5% of autooxidation products or purified cholesterol. Lipid accumulation took place in the liver and intramural myocardial vessels during feeding of rabbits with autooxidated cholesterol. Feeding with pure cholesterol free from autooxidation products caused but slight changes or no changes at all.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

Gas chromatography of bile acids

Bile acids, the end products of cholesterol metabolism in the liver, are of vital importance in the tissue distribution of cholesterol. Abnormalities in cholesterol biosynthesis or metabolism are often reflected in the proportions, concentrations and conjugation of bile acids in various tissues and determination of bile acids in these tissues is important in the diagnosis of hepatobiliary diseases. Several methods for quantitative determination of bile acids in biological fluids are known and have been reviewed. In this review, we have discussed the gas-chromatographic method for determination of bile acids with special reference to bile acid quantitation in plasma, bile, urine and stool.     

Aminoguanidine and metformin prevent the reduced rate of HDL-mediated cell cholesterol efflux induced by formation of advanced glycation end products

OBJECTIVE: The mechanisms whereby advanced glycation end products (AGE) contribute to atherogenesis in diabetes mellitus are not fully understood. In this study we analyzed in vitro the influence of advanced glycated albumin (AGE-albumin) as well as the role of the AGE inhibitors--aminoguanidine (AMG) and metformin (MF)--on the cell cholesterol efflux. METHODS: HDL3 and albumin-mediated cholesterol efflux was measured in mouse peritoneal macrophages and in SR-BI transfected cells that had been treated along time with dicarbonyl sugars or AGE-albumin, both in the presence or in the absence of AMG and MF. 125I-HDL3 cell binding and 125I-AGE-albumin cell degradation were measured. Carboxymethyllysine (CML) formation and SR-BI expressions were determined by immunoblot. RESULTS: AGE-albumin efficiently trapped cell cholesterol but impaired the HDL-mediated cell cholesterol efflux by decreasing HDL binding to the cell surface and inducing intracellular glycoxidation, without interfering with the SR-BI expression. Cell treatment with dicarbonyl sugars also disrupted the HDL-mediated cell cholesterol efflux, but this was prevented by AMG and MF that reduced CML formation. CONCLUSIONS: By adversely impairing the HDL-mediated cell cholesterol removal rate, AGE-albumin and cell glycoxidation could facilitate the development of premature atherosclerosis in diabetes mellitus (DM) and in other diseases associated with carbonyl and oxidative stress like in chronic uremia. Thus, drugs that prevent AGE formation may be useful to correct disturbances in cell cholesterol transport.     

Lipid regulation of cell membrane structure and function

Recent studies of structure-function relationships in biological membranes have revealed fundamental concepts concerning the regulation of cellular membrane function by membrane lipids. Considerable progress has been made in understanding the roles played by two membrane lipids: cholesterol and phosphatidyl-ethanolamine. Cholesterol has been shown to regulate ion pumps, which in some cases show an absolute dependence on cholesterol for activity. These studies suggest that an essential role that cholesterol plays in mammalian cell biology is to enable crucial membrane enzymes to provide function necessary for cell survival. Studies of phosphatidylethanolamine regulation of membrane protein activity and regulation of membrane morphology led to hypotheses concerning the roles for this particular lipid in biological membranes. New information on lipid-protein interactions and on the nature of the lipid head groups has permitted the development of mechanistic hypotheses for the regulation of membrane protein activity by phosphatidyl-ethanolamine. In addition, intermediates in the lamellar-nonlamellar phase transitions of membrane systems containing phosphatidylethanolamine, or other lipids with similar properties, have recently been implicated in facilitating membrane fusion. Finally, studies of transmembrane movement of lipids have provided new insight into the regulation of membrane lipid asymmetry and the biogenesis of cell membranes. These kinds of studies are harbingers of a new generation of progress in the field of cell membranes.     

Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes

Enlarged fat cells exhibit modified metabolic capacities, which could be involved in the metabolic complications of obesity at the whole body level. We show here that sterol regulatory element-binding protein 2 (SREBP-2) and its target genes are induced in the adipose tissue of several models of rodent obesity, suggesting cholesterol imbalance in enlarged adipocytes. Within a particular fat pad, larger adipocytes have reduced membrane cholesterol concentrations compared with smaller fat cells, demonstrating that altered cholesterol distribution is characteristic of adipocyte hypertrophy per se. We show that treatment with methyl-beta-cyclodextrin, which mimics the membrane cholesterol reduction of hypertrophied adipocytes, induces insulin resistance. We also produced cholesterol depletion by mevastatin treatment, which activates SREBP-2 and its target genes. The analysis of 40 adipocyte genes showed that the response to cholesterol depletion implicated genes involved in cholesterol traffic (caveolin 2, scavenger receptor BI, and ATP binding cassette 1 genes) but also adipocyte-derived secretion products (tumor necrosis factor alpha, angiotensinogen, and interleukin-6) and proteins involved in energy metabolism (fatty acid synthase, GLUT 4, and UCP3). These data demonstrate that altering cholesterol balance profoundly modifies adipocyte metabolism in a way resembling that seen in hypertrophied fat cells from obese rodents or humans. This is the first evidence that intracellular cholesterol might serve as a link between fat cell size and adipocyte metabolic activity.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function

A spectrum of cholesterol oxidation derivatives (oxysterols) is generated in food products exposed to heat or radiation in the presence of oxygen. One of these derivatives (cholestan-3 beta,5 alpha,6 beta-triol) was shown to compromise the selective barrier function of cultured vascular endothelial cell monolayers, an action that may initiate atherosclerotic lesion formation. This study sought to investigate the relationship of cholesterol synthesis inhibition by several naturally occurring oxysterols to depression of vascular endothelial cell monolayer barrier function, determined as an increase in albumin transfer across cultured endothelial monolayers. All oxysterols tested caused a variable time- and dose-dependent elevation in trans-endothelial albumin transfer, and they were also able to inhibit cholesterol biosynthesis to varying degrees. Pure cholesterol was without effect on both counts. The correlation between the increase in albumin transfer related to oxysterol exposure and the ability of oxysterols to suppress cholesterol biosynthesis was, however, poor. Moreover, mevinolin, a water-soluble competitive inhibitor of cholesterol synthesis, reduced the rate of cholesterol synthesis to 0.9% of control but did not significantly increase albumin transfer. Cholestan-3 beta,5 alpha,6 beta-triol caused a 660% elevation in albumin transfer while cholesterol synthesis remained at 11% of control. We conclude that changes in endothelial barrier function caused by exposure to the oxysterols examined, but not pure cholesterol, are probably related to factors other than the well-known action of cholesterol biosynthesis inhibition. These findings may have implications in the development of atherosclerosis.     

Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The cholesterol content of human erythrocyte membranes has been modified by incubation of intact cells with sonicated egg phosphatidylcholine/cholesterol vesicles and with egg phosphatidylcholine vesicles. (Na+ + K+)-ATPase ATP hydrolyzing activity was measured as a function of membrane cholesterol content. High membrane cholesterol inhibits the ATPase activity of the enzyme and low membrane cholesterol activates that enzyme activity. The most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation. Ouabain binding studies show that the inhibition is not due to a loss of enzyme from the membrane.     

Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2

The human hepatoma cell line Hep-G2 has been shown to express the major enzymes of intra- and extracellular cholesterol metabolism. These include lecithin:cholesterol acyltransferase, acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-7 alpha-hydroxylase. Regulatory mechanisms that have been described in other hepatic systems also appear to be active in Hep-G2 cells: perturbations of cholesterol and triglyceride metabolism affected the enzyme activities and the accumulation of specific apolipoproteins in the culture media. The results indicate that studies of Hep-G2 cells may provide useful information for the elucidation of mechanisms of regulation of human hepatocyte cholesterol, lipoprotein, and biliary metabolism.