Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

Ruan, Benfang; Shey, Justin; Gerst, Nicolas; Wilson, William K.; Schroepfer, George J.

doi:10.1073/pnas.93.21.11603

Cited by 31 publications

(26 citation statements)

References 67 publications

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“…It is obvious from our experiments that it is very difficult to resolve the biosynthetic products deriving from acetate with TLC-based methods only. This is true especially for the other C27 sterols, i.e., immediate precursors of cholesterol, and in accordance with the report of Ruan et al (27). The transport of biosynthetic sterol intermediates is known to differ from that of the end product (39), and pooling of the sterol precursors together with cholesterol therefore will disturb the analysis.…”

Section: Discussionsupporting

confidence: 68%

“…Newly synthesized cholesterol was detected by pulse labeling the cells with [ 3 H]acetate, extracting the cellular material in organic solvent, and analyzing it by TLC followed by HPLC on a silver-ion column (Ag ϩ -HPLC). This method separates sterols based on the number and position of double bonds (27,28) and resolves even the immediate precursor desmosterol from cholesterol ( Fig. 1a).…”

Section: Rate Of Cholesterol Synthesis In Bhk Cellsmentioning

confidence: 99%

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Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Heino

Lusa

Somerharju

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

230

193

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In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

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Section: Discussionsupporting

confidence: 68%

Section: Rate Of Cholesterol Synthesis In Bhk Cellsmentioning

confidence: 99%

Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Heino

Lusa

Somerharju

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

230

193

View full text Add to dashboard Cite

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“…FF-MAS and 14␣-ethyl-5␣-cholest-7-ene-3␤,15␣-diol were synthesized as previously described [14,15]. The FF-MAS samples showed no sterol impurities in the 500-MHz NMR spectra (1% detection limit).…”

Section: Sterols and Other Chemicalsmentioning

confidence: 99%

Meiosis-Activating Sterol and the Maturation of Isolated Mouse Oocytes1

Downs

Ruan

Schroepfer

2001

Biology of Reproduction

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This study was carried out to examine the effects of the meiosis-activating C(29) sterol, 4,4-dimethyl-5 alpha-cholesta-8,14, 24-trien-3 beta-ol (FF-MAS), on mouse oocyte maturation in vitro. Cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) from hormonally primed, immature mice were cultured 17-18 h in minimum essential medium (MEM) containing 4 mM hypoxanthine plus increasing concentrations of FF-MAS. The sterol induced maturation in DO with an optimal concentration of 3 microg/ml but was without effect in CEO, even at concentrations as high as 10 microg/ml. Some stimulation of maturation in hypoxanthine-arrested CEO was observed when MEM was replaced by MEMalpha. Interestingly, the sterol suppressed the maturation of hypoxanthine-arrested CEO in MEM upon removal of glucose from the medium. FF-MAS also failed to induce maturation in DO when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP). The rate of maturation in FF-MAS-stimulated, hypoxanthine-arrested DO was slow, as more than 6 h of culture elapsed before significant meiotic induction was observed, and this response required the continued presence of the sterol. Although the oocyte took up radiolabeled lanosterol, such accumulation was restricted by the presence of cumulus cells. In addition, lanosterol failed to augment FSH-induced maturation and was even inhibitory at a high concentration. Moreover, the downstream metabolite, cholesterol, augmented the inhibitory action of dbcAMP on maturation in both CEO and DO. Two inhibitors of 14 alpha-demethylase, ketoconazole, and 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol that can suppress FF-MAS production from lanosterol failed to block consistently FSH-induced maturation. These results confirm the stimulatory action of FF-MAS on hypoxanthine-arrested DO but do not support a universal meiosis-inducing function for this sterol.

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“…Further purification was achieved on a silver column prepared according to the method described by Ruan et al [20] and was eluted with hexane/ 9.1% acetone. Lanosterol and FF-MAS were separated with an initial elution at 1 ml/min, which brought off lanosterol at approximately 11-12 min.…”

Section: Hplc Analysesmentioning

confidence: 99%

Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis?1

Cao

Pomerantz

Popliker

et al. 2004

Biology of Reproduction

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Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

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Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

Cited by 31 publications

References 67 publications

Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface

Meiosis-Activating Sterol and the Maturation of Isolated Mouse Oocytes1

Meiosis-Activating Sterol Synthesis in Rat Preovulatory Follicle: Is It Involved in Resumption of Meiosis?1

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