Cholesterol Journal Articles

Record 7081 to 7100

Identification of a scavenger receptor in rat luteal cells which recognizes chemically modified lipoproteins and mediates the uptake of cholesterol for steroidogenesis
Chen, Z. and K. M. Menon (1993), Biochim Biophys Acta 1150(1): 79-88.
Abstract: The presence of the acetyl low density lipoprotein (acetyl-LDL), or scavenger receptor, which binds modified forms of LDL, was examined in rat luteal cells. Acetyl-LDL supported progesterone production by dispersed rat luteal cells at least equal to that of the native LDL under basal conditions and in the presence of human chorionic gonadotropin (hCG). The acetyl-LDL-supported progesterone production was stimulated by hCG and dibutyryl cAMP in a concentration-dependent manner. Studies on acetyl-LDL binding to ovarian plasma membranes revealed a single class of binding sites with high affinity. The binding is specific in that unlabeled acetyl-LDL and fucoidin, a known competitor for binding to scavenger receptor, were effective competitors, while native LDL was not. Furthermore, degradation of 125I-acetyl-LDL by cultured luteal cells was inhibited by unlabeled acetyl-LDL and fucoidin but not native LDL. The experiments on cross-competition between acetyl-LDL and tetranitromethane-modified high density lipoprotein (TNM-HDL) indicated that TNM-HDL is also recognized as a ligand by this receptor. In addition, in vivo pretreatment of rats with hCG resulted in induction of acetyl-LDL binding activity of ovarian plasma membranes in a time-dependent manner when compared to saline injected controls. The increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, this study demonstrates that, in addition to receptor-mediated LDL and HDL pathways, rat luteal cells possess a scavenger receptor pathway, which recognizes TNM-HDL as well as acetyl-LDL. This receptor may play an important role in the uptake and utilization of modified lipoprotein-associated cholesterol by luteal cells.

Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles
Sviridov, D., L. Pyle, et al. (1996), Biochemistry 35(1): 189-96.
Abstract: The effect of monoclonal antibodies against apolipoprotein A-I (apoA-I) on the efflux of intracellular and plasma membrane cholesterol from HepG2 cells to human serum, high-density lipoprotein (HDL), apoA-I, and apoA-I/phosphatidylcholine complex (apoA-I/PC) was studied. Fab fragments of two monoclonal antibodies, AI-3 (residues 140-147) and Al-4.2 (residues 149-150), inhibited the efflux of intracellular cholesterol to serum in a dose-dependent manner. In combination, these antibodies were twice as effective than when used alone. None of the antibodies tested inhibited efflux of the plasma membrane cholesterol. When different types of acceptors were compared for their ability to promote intracellular cholesterol efflux, they were effective in the following order: serum > HDL > apoA-I/PC > pure apoA-I. Antibody AI-3 inhibited efflux of intracellular cholesterol to serum, HDL, and pure apoA-I, but not to apoA-I/PC. Antibody AI-4.2 inhibited efflux to serum, apoA-I/PC, and pure apoA-I, but not to HDL. An explanation for this is that antibody AI-4.2 reacts poorly with isolated alpha-HDL in an immunoprecipitation assay and has higher affinity for pre beta 2-HDL and pre beta 3-HDL particles than antibody AI-3 in nondenaturing two-dimensional electrophoresis. In conclusion, we have demonstrated that a region of apoA-I within or adjacent to residues 140-150 determines the ability of apoA-I to promote intracellular cholesterol efflux.

Identification of an apolipoprotein A-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1
Natarajan, P., T. M. Forte, et al. (2004), J Biol Chem 279(23): 24044-52.
Abstract: Synthetic peptides were used in this study to identify a structural element of apolipoprotein (apo) A-I that stimulates cellular cholesterol efflux and stabilizes the ATP binding cassette transporter A1 (ABCA1). Peptides (22-mers) based on helices 1 (amino acids 44-65) and 10 (amino acids 220-241) of apoA-I had high lipid binding affinity but failed to mediate ABCA1-dependent cholesterol efflux, and they lacked the ability to stabilize ABCA1. The addition of helix 9 (amino acids 209-219) to either helix 1 (creates a 1/9 chimera) or 10 (9/10 peptide) endowed cholesterol efflux capability and ABCA1 stabilization activity similar to full-length apoA-I. Adding helix 9 to helix 1 or 10 had only a small effect on lipid binding affinity compared with the 22-mer peptides, indicating that helix length and/or determinants on the polar surface of the amphipathic alpha-helices is important for cholesterol efflux. Cholesterol efflux was specific for the structure created by the 1/9 and 9/10 helical combinations, as 33-mers composed of helices 1 and 3 (1/3), 2/9, and 4/9 failed to mediate cholesterol efflux in an ABCA1-dependent manner. Transposing helices 9 and 10 (10/9 peptide) did not change the class Y structure, hydrophobicity, or amphiphilicity of the helical combination, but the topography of negatively charged amino acids on the polar surface was altered, and the 10/9 peptide neither mediated ABCA1-dependent cholesterol efflux nor stabilized ABCA1 protein. These results suggest that a specific structural element possessing a linear array of acidic residues spanning two apoA-I amphipathic alpha-helices is required to mediate cholesterol efflux and stabilize ABCA1.

Identification of apoprotein A-1 in pure cholesterol gallstone
Saito, H., H. Nishimura, et al. (1991), Nippon Shokakibyo Gakkai Zasshi 88(9): 2186.

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter
Kramer, W., H. Glombik, et al. (2000), FEBS Lett 487(2): 293-7.
Abstract: To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M(r) 145.3+/-7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M(r) 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.

Identification of candidate genes regulating HDL cholesterol using a chromosomal region expression array
Cox, L. A., S. Birnbaum, et al. (2002), Genome Res 12(11): 1693-702.
Abstract: To identify candidate genes encoding QTLs in baboons, we have developed a novel strategy that integrates comparative mapping, bioinformatics, and expression arrays. A genome-wide scan, performed previously on pedigreed baboons to localize QTLs for phenotypes that are known risk factors for atherosclerosis, revealed a QTL on chromosome 18q that influences high-density lipoprotein cholesterol (HDL-C) phenotypes. After ruling out the only two biologically relevant positional candidate genes in this chromosomal region, we combined information from baboon pedigrees and HDL-C phenotypes with a baboon microsatellite marker map, human microsatellite marker maps, and human genome maps to develop a chromosomal region expression array (CREA). The CREA was screened with heterologous liver cDNA from sib-pairs of contrasting HDL-C phenotypes on two different diets, and genes were prioritized for further study by expression profiles. Analysis of gene expression in this restricted chromosomal region, combined with HDL-C phenotypic information, yielded a list of candidate genes for the QTL regulating HDL-C in baboons. Our data demonstrate the power of this strategy for identifying candidate genes encoding QTLs for multigenic traits. This strategy is applicable to many species that serve as models for human diseases and can even be used with human subjects.

Identification of cholelithogenic enterohepatic helicobacter species and their role in murine cholesterol gallstone formation
Maurer, K. J., M. M. Ihrig, et al. (2005), Gastroenterology 128(4): 1023-33.
Abstract: BACKGROUND & AIMS: Helicobacter spp are common inhabitants of the hepatobiliary and gastrointestinal tracts of humans and animals and cause a variety of well-described diseases. Recent epidemiologic results suggest a possible association between enterohepatic Helicobacter spp and cholesterol cholelithiasis, chronic cholecystitis, and gallbladder cancer. To test this, we prospectively investigated the effects of Helicobacter spp infection in cholesterol gallstone pathogenesis in the highly susceptible C57L/J mouse model. METHODS: Helicobacter spp-free adult male C57L mice were infected with several different enterohepatic Helicobacter spp or left uninfected and fed either a lithogenic diet or standard mouse chow for 8 and 18 weeks. At the conclusion of the study, bile was examined microscopically and diagnostic culture and polymerase chain reaction were performed. RESULTS: Mice infected with Helicobacter bilis or coinfected with Helicobacter hepaticus and Helicobacter rodentium and fed a lithogenic diet developed cholesterol gallstones at 80% prevalence by 8 weeks compared with approximately 10% in uninfected controls. Monoinfections with H hepaticus, Helicobacter cinaedi, and H rodentium gave a cholesterol gallstone prevalence of 40%, 30%, and 20%, respectively; the latter 2 groups did not differ significantly from uninfected animals. Neither infected nor uninfected mice fed a chow diet developed cholesterol gallstones. CONCLUSIONS: These findings, along with prior epidemiologic studies, suggest that Helicobacter spp play a major role in the pathophysiology of cholesterol gallstone formation in mice and perhaps humans.

Identification of cholesterol esters using topological criteria
Liapkov, B. G. and D. I. Voinov (1990), Vopr Med Khim 36(5): 85-8.
Abstract: Cholesterol esters obtained from biological sources were separated using highly-effective liquid chromatography. Identification of individual molecular forms of these esters was carried out by means of linear correlation between the period of their retaining and the topological indices by Balaban and Randich, describing the structure of descriptors (acyl group) in cholesterol esters.

Identification of cholesterol-bound aldehydes in copper-oxidized low density lipoprotein
Kamido, H., A. Kuksis, et al. (1992), FEBS Lett 304(2-3): 269-72.
Abstract: Lipid-soluble cholesteryl ester core aldehydes (aldehydes still bound to the cholesterol ring) were identified among the products of copper-catalyzed peroxidation of human low density lipoprotein (LDL). The LDL was exposed to oxygenated buffer and 5 microM CuSO4 for 24 h. The core aldehydes were isolated as the dinitrophenylhydrazones, and were identified by reverse-phase HPLC with mass spectrometry. The major components were the C4-C10 oxoalkanoyl esters of cholesterol and 7-ketocholesterol, and accounted for 1-2% of the cholesteryl linoleate and arachidonate consumed.

Identification of genetic variants in endothelial lipase in persons with elevated high-density lipoprotein cholesterol
deLemos, A. S., M. L. Wolfe, et al. (2002), Circulation 106(11): 1321-6.
Abstract: BACKGROUND: Elevated high-density lipoprotein cholesterol (HDL-C) is associated with reduced risk of cardiovascular disease, and variation in HDL-C levels has been shown to be approximately 50% heritable. Overexpression of endothelial lipase (EL), a member of the lipoprotein lipase gene family, markedly reduces HDL-C levels in mouse models. We hypothesized that genetic variation in EL might be associated with elevated HDL-C. METHODS AND RESULTS: All exons and 1.2 kilobase of promoter of the EL gene were sequenced in 20 unrelated human subjects with high HDL-C levels. A total of 17 variants were identified. Six of these were potentially functional and were confirmed by restriction enzyme analysis. Four variants result in amino acid changes (Gly26Ser, Thr111Ile, Thr298Ser, and Asn396Ser) and 2 variants were in the promoter (-303A/C and -410C/G). The genotype frequencies of each variant were determined in 176 black controls, 165 white controls, and 123 whites with high HDL-C. The Thr111Ile variant was the most common, with an allele frequency of 10.3% in blacks, 31.2% in white controls, and 32.6% in the high HDL-C group. The remaining variants all had allele frequencies <5.0% but differed in frequency among the 3 groups. Interestingly, Gly26Ser, Thr298Ser, and -303A/C were found in the black and high HDL-C white cohorts but were absent in the control white group. CONCLUSIONS: Six new potentially functional variants in EL were discovered through sequencing of the EL gene in subjects with high HDL-C levels. Differences in allele frequencies exist between blacks and whites and between control subjects and those with high HDL-C levels.

Identification of Helicobacter pylori DNA in human cholesterol gallstones
Monstein, H. J., Y. Jonsson, et al. (2002), Scand J Gastroenterol 37(1): 112-9.
Abstract: BACKGROUND: The gallbladder mucosa secretes hydrogen ions and is covered by mucus. The environmental conditions for bacterial colonization are similar to those in the stomach. Gallbladder stones often contain DNA from enteric bacteria, but no compelling evidence demonstrates that Helicobacter spp. have been present. The aim of this study was to establish bacterial DNA profiles in cholesterol gallstones with special reference to Helicobacter pylori. METHODS: Cholesterol gallstones from 20 patients were subjected to polymerase chain reaction, bacterial profiling by temporal temperature gradient gel electrophoresis, automated DNA sequencing, and Southern blot analysis using a Helicobacter sp. specific primer. A nested ureI-PCR assay was used to discriminate between gastric and non-gastric H. pylori. RESULTS: TTGE, partial 16S rDNA sequencing, and hybridization analysis revealed the presence of DNA presumably representing a mixed bacterial flora in cholesterol gallstones, including H. pylori in the gallstone centres in 11 out of 20 patients. In three cases, the urel-PCR assay revealed non-gastric H. pylori. CONCLUSIONS: These data support the presence of DNA from a mixed bacterial population, including H. pylori in cholesterol gallstones, reflecting either that H. pylori is an indigenous part of a flora in the stone-containing gallbladder or, alternatively, that H. pylori colonization in the biliary tract predisposes to cholesterol gallstone formation.

Identification of human biliary alpha 1-acid glycoprotein as a cholesterol crystallization promoter
Abei, M., H. Nuutinen, et al. (1994), Gastroenterology 106(1): 231-8.
Abstract: BACKGROUND/AIMS: We have recently outlined the biochemical features of a human 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. The goal in this work was to establish its identity and to examine some aspects of its biochemical properties relative to its activity. METHODS: Internal amino acid sequencing following tryptic digestion was performed. Based upon this result, immunoreactivity against the 42-kilodalton glycoprotein was examined using a relevant antibody. With the same antibody, the 42-kilodalton glycoprotein was isolated from bile and assayed for activity. Sequential enzymatic deglycosylation of successive terminal glycans of the purified glycoprotein was performed, and the effects on both reductions in molecular radius (M(r)) and on comparative promoter activities were examined. RESULTS: Both amino acid sequence and immunochemical data identify the 42-kilodalton glycoprotein as a biliary form of alpha 1-acid glycoprotein. When purified by immunoaffinity chromatography, potent promoting activity shown was proportionately reduced by successive removal of terminal glycans that also reduced the M(r)s. CONCLUSIONS: The 42-kilodalton cholesterol crystallization-promoting glycoprotein is now identified as a biliary form of alpha 1-acid glycoprotein. Further, some aspects of the important role of glycans in this extensively glycosylated protein have been explored.

Identification of impaired cholesterol biosynthesis in a Japanese patient of Smith-Lemli-Opitz syndrome
Hasui, M., C. Saito, et al. (1997), No To Hattatsu 29(1): 61-6.
Abstract: We reported the decreased level of cholesterol as well as the elevated levels of 7- and 8-dehydrocholesterol in the serum and erythrocytes of a Japanese patient with Smith-Lemli-Opitz syndrome. These findings suggested that the detection of these precursors of cholesterol synthesis should become an important biochemical parameter for this syndrome in which clinical features are not always obvious.

Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo
Hashimoto, F. and H. Hayashi (1991), Biochim Biophys Acta 1086(1): 115-24.
Abstract: Cholesterol synthesis from mevalonate is inhibited by aminotriazole treatment in vivo. We tried to identify intermediates accumulated in liver of aminotriazole-treated rats. At 6 h after the aminotriazole treatment, the liver was excised. Sterols were extracted from it, and subjected to capillary gas-liquid chromatography, high-performance liquid chromatography, gas-liquid chromatography linked to mass spectrometry and gas-liquid chromatography linked to Fourier-transform infrared spectrometry. It was found that 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest- 8-en-3 beta-ol were accumulated in the liver, mainly as the free forms. The contents of the former and the latter were increased to 25- and 64-times the control values, respectively. In another experiment, 2-13Cmevalonate was injected at 2 h after aminotriazole treatment, and 4 h later the liver was excised. The sterols extracted from the liver were subjected to gas-liquid chromatography linked to mass spectrometry. Specific fragment ions reflecting the incorporation of 13C mevalonate were detected in the mass spectra of the intermediate sterols. Accumulation of 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol after aminotriazole treatment suggests that elimination of the 4 alpha-methyl group from 4-methyl intermediate sterols is inhibited by aminotriazole.

Identification of intermediates in the conversion of cholesterol to pregnenolone with a reconstituted cytochrome p-450scc system: accumulation of the intermediate modulated by the adrenodoxin level
Sugano, S., R. Miura, et al. (1996), J Biochem (Tokyo) 120(4): 780-7.
Abstract: Dihydroxycholesterol and pregnenolone were clearly detected on HPLC when 22R-hydroxycholesterol was incubated with a reconstituted P450scc system containing equimolar amounts of P450scc and adrenodoxin. The dihydroxycholesterol, which has been accepted to be an intermediate in the conversion of 22R-hydroxycholesterol to pregnenolone, accumulated when adrenodoxin was at a subsaturating level with respect to P450scc. The formation of the intermediate increased with increasing pH in the range of 7.2 to 8.1, and the ratio of the intermediate to the product, pregnenolone, increased with increasing pH. When the binding of P450scc to adrenodoxin was weakened by elevation of the ionic strength, the formation of the intermediate relative to the product increased. The apparent Km for dihydroxycholesterol at a subsaturating level of adrenodoxin was about 7 microM, in contrast to 4 microM at a saturating level of adrenodoxin, implying that the affinity of dihydroxycholesterol is lower at a subsaturating level of adrenodoxin than at a saturating one. These results suggest that a subsaturating level of adrenodoxin weakened the binding of dihydroxycholesterol to P450scc and thus the intermediate, dihydroxycholesterol, was released. An intermediate other than dihydroxycholesterol, obtained when cholesterol was used as the substrate, was identified as 22R-hydroxycholesterol by HPLC and mass spectroscopic analysis. The intermediate obtained when 22R-hydroxycholesterol was used as the substrate was identified as 20R,22R-dihydroxycholesterol by HPLC, mass, and 1H-NMR spectroscopic analyses. These results provide direct evidence that cholesterol is metabolized to pregnenolone by way of 22R-hydroxycholesterol and 20R,22R-dihydroxycholesterol by P45 scc.

Identification of novel differentially expressed hepatic genes in cholesterol-fed rabbits by a non-targeted gene approach
Remaley, A. T., U. K. Schumacher, et al. (1995), J Lipid Res 36(2): 308-14.
Abstract: Several key genes involved in cholesterol metabolism are known to be directly regulated by cholesterol. The possible indirect effect, however, of increased levels of cellular cholesterol on gene expression and its possible role in cholesterol metabolism and atherosclerosis has not been thoroughly explored. In order to determine the overall effect of cholesterol on gene expression, we isolated differentially expressed genes from a PCR-based subtraction library prepared from the liver of chow-fed and cholesterol-fed rabbits. A total of nine upregulated and four down-regulated cDNA fragments were isolated. As determined by Northern blot analysis, the expression of the isolated cDNAs began to change as early as the first week on the cholesterol-rich diet or as late as 4 weeks, which corresponded with hepatic cholesterol accumulation. Three of the cDNAs were identified by DNA sequence homology, whereas the remaining cDNAs had no significant homology match. CYP1A1, a cytochrome P450 isoenzyme, was found to be down-regulated in hepatocytes by cholesterol feeding. Osteopontin and Mac-2, which are produced by macrophages, were found to be up-regulated in Kupffer cells by cholesterol feeding. Overall these results demonstrate the usefulness of the subtraction library approach for identifying new candidate genes for exploring the pathogenesis of atherosclerosis.

Identification of quantitative trait loci for serum cholesterol levels in stroke-prone spontaneously hypertensive rats
Kato, N., T. Tamada, et al. (2000), Arterioscler Thromb Vasc Biol 20(1): 223-9.
Abstract: The stroke-prone spontaneously hypertensive rat (SHRSP) has been reported to show significantly lower levels of serum total cholesterol than the normotensive control strain Wistar-Kyoto rat (WKY). Because selective inbreeding was conducted for stroke proneness, this concomitantly inherited characteristic of SHRSP may play some pathophysiological role in stroke. We evaluated the genetic determinants of the cholesterol trait by estimating heritability and subsequently by undertaking a genome-wide screen with 161 genetic markers in F(2) progeny involving SHRSP and WKY (104 male and 106 female rats). Three quantitative trait loci (QTLs) were detected on rat chromosomes 5, 7, and 15. Markers from the linked region on chromosome 15 indicated significant evidence of linkage with a maximal log of the odds (LOD) score of 7.7, whereas those on chromosomes 5 and 7 cosegregated with the trait in a sex-specific manner (the QTL close to genetic marker D5 Mit5 reached an LOD score of 7.3 in males, and that close to D7 Mit10 reached an LOD score of 3.2 in females). The male-specific QTL on chromosome 5 appeared to overlap with previously reported QTLs for stroke-associated phenotypes, but an identical gene (or genes) appeared unlikely to control these and the cholesterol traits simultaneously. In the present study, serum cholesterol levels were shown to be highly genetically determined in SHRSP (the heritability estimates are 76% in males and 83% in females), and 3 QTLs with substantial effects were identified. Further work, however, is required to clarify whether the cholesterol trait is related to the etiology of stroke or has been retained by chance through the inbreeding process in SHRSP.

Identifying adults at increased risk of coronary disease. How well do the current cholesterol guidelines work?
Grover, S. A., L. Coupal, et al. (1995), Jama 274(10): 801-6.
Abstract: OBJECTIVE--To assess the accuracy of lipid screening strategies to identify individuals at increased risk of coronary heart disease mortality. PATIENTS--The 15% random sample of adults recruited into the Lipid Research Clinic Prevalence and Follow-up Studies, which included 3678 men and women aged 35 to 74 years. Total plasma cholesterol levels, lipoprotein fractions, and other coronary risk factors at study entry were compared with subsequent coronary heart disease mortality (mean follow-up, 12.2 years). MAIN OUTCOME MEASURES--The areas under receiver operating characteristic curves for blood lipids, lipid ratios, the screening guidelines proposed by the National National Cholesterol Education Program, those of the Canadian Consensus Conference on Cholesterol, and a coronary risk model that used Framingham data. MAIN RESULTS--The current National Cholesterol Education Program guidelines (area under the curve, 0.74) were significantly (P =.03) more accurate than the old National Cholesterol Education Program guidelines (area, 0.72). The ratio of total plasma cholesterol level to high-density lipoprotein cholesterol level (area, 0.72) was as accurate as current National Cholesterol Education Program guidelines. The coronary risk model (area, 0.85) was superior (P <.003) to all other screening maneuvers. Compared with the current National Cholesterol Education Program guidelines, the risk model demonstrated superior test sensitivity (70% vs 45%) with only slightly reduced specificity (82% vs 86%). CONCLUSION--The ratio of total plasma cholesterol level to high-density lipoprotein cholesterol level is as accurate as current American screening guidelines. Future guidelines should better incorporate high-density lipoprotein cholesterol levels and nonlipid risk factors to target high-risk individuals accurately.

Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with 125Iazido-cholesterol
Corbin, J., H. H. Wang, et al. (1998), Biochim Biophys Acta 1414(1-2): 65-74.
Abstract: A novel photoreactive analog of cholesterol, 3alpha-(4-azido-3-125Iiodosalicylic)-cholest-5-ene (125Iazido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated 125Iazido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of 125Iazido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alphaV8-20 (alphaSer-173-Glu-338), alphaV8-10 (alphaAsn-339-Gly-439), and gammaV8-14 (gammaLeu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. 125IAzido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR.

Identity of the cholesterol-raising factor from boiled coffee and its effects on liver function enzymes
Weusten-Van der Wouw, M. P., M. B. Katan, et al. (1994), J Lipid Res 35: 721-33.
Abstract: Boiled coffee contains an unidentified lipid that raises serum cholesterol. We studied the effects of the ingestion of coffee oil fractions of increasing purity in volunteers in order to identify the cholesterol-raising factor. In 15 volunteers who ingested 0.75 g/d of a non-triglyceride-fraction from coffee oil for 4 weeks, mean cholesterol increased by 48 mg/dl (1.2 mmol/l) relative to placebo. In contrast, a coffee oil stripped of the non-triglyceride lipids cafestol and kahweol had no effect. In three volunteers, purified cafestol (73 mg/d) plus kahweol (58 mg/d) increased cholesterol by 66 mg/dl (1.7 mmol/l) after 6 weeks. Oil from Robusta beans, which contains cafestol but negligible kahweol, also raised serum cholesterol. These findings show that cafestol is at least partly responsible for the cholesterol-raising effect of boiled coffee. Coffee oils and brews containing cafestol consistently increased serum triglycerides and alanine amino-transferase, and depressed serum creatinine and gamma-glutamyl-transferase (GGT). After withdrawal GGT activity rose above baseline. Norwegians who habitually consumed 5-9 cups of boiled coffee per day had higher serum cholesterol levels and lower GGT but no higher alanine aminotransferase activity than controls. Thus, serum cholesterol is raised by cafestol and possibly also kahweol, both natural components of coffee beans. The mechanism of action is unknown but is accompanied by alterations in liver function enzymes.

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