Rapid analysis of oxidized cholesterol derivatives by high‐performance liquid chromatography combined with diode‐array ultraviolet and evaporative laser light‐scattering detection

Osada, Kyoichi; Ravandi, Amir; Kuksis, A.

doi:10.1007/s11746-999-0078-3

Cited by 21 publications

(13 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…17 The bottom chloroform layer was then washed three times with a methanol-water mixture (5:4, v/v), collected, and solvent removed using a SpeedVac. The cholesterol level was measured using HPLC as recently described.…”

Section: Methodsmentioning

confidence: 99%

Hexavalent Chromium-induced Alteration of Proteomic Landscape in Human Skin Fibroblast Cells

2013

View full text Add to dashboard Cite

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K2Cr2O7). We were able to quantify ~ 4600 unique proteins, among which ~ 400 exhibited significant alterations in expression levels upon a 24-hr treatment with 0.5 μM K2Cr2O7. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K2Cr2O7 treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K2Cr2O7 treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells.

show abstract

Section: Methodsmentioning

confidence: 99%

Hexavalent Chromium-induced Alteration of Proteomic Landscape in Human Skin Fibroblast Cells

2013

View full text Add to dashboard Cite

show abstract

“…The bottom layer was then collected, placed in a test tube, and evaporated to dryness with dry N 2 gas. The cholesterol level was determined by HPLC using methanol as mobile phase, and a UV detector, with the wavelength of detection being set at 205 nm, was employed for monitoring the effluent26.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Proteomic Analysis Reveals the Perturbation of Multiple Cellular Pathways in Jurkat-T Cells Induced by Doxorubicin

et al. 2010

View full text Add to dashboard Cite

Doxorubicin remains an important part of chemotherapy regimens in the clinic and is considered as an effective agent in the treatment of acute lymphoblastic leukemia (ALL). Although the cellular responses induced by doxorubicin treatment have been investigated for years, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we utilized mass spectrometry, together with stable isotope labeling by amino acids in cell culture (SILAC), to analyze comparatively the protein expression in Jurkat-T cells before and after treatment with a clinically relevant concentration of doxorubicin. We were able to quantify 1066 proteins in Jurkat-T cells with both forward and reverse SILAC measurements, among which 62 were with significantly altered levels of expression induced by doxorubicin treatment. These included the upregulation of core histones, heterogeneous nuclear ribonucleoproteins and superoxide dismutase 2 as well as the down-regulation of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase. The latter two are essential enzymes for cholesterol biosynthesis. We further demonstrated that the doxorubicin-induced growth inhibition of Jurkat-T cells could be rescued by treatment with cholesterol, supporting that doxorubicin exerts its cytotoxic effect, in part, via suppressing the expression of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase, thereby inhibiting the endogenous production of cholesterol. The results from the present study provided important new knowledge for gaining insights into the molecular mechanisms of action of doxorubicin.

show abstract

“…Detailed mass spectral and retention data on sterol oxides are available to support identification 132. 178 HPLC, widely used in the analysis of cholesterol oxides,181, 182 is not effective enough to separate the large number of plant sterol products.…”

Section: Methodsmentioning

confidence: 99%

Plant sterols: biosynthesis, biological function and their importance to human nutrition

Piironen

Lindsay

Miettinen

et al. 2000

J. Sci. Food Agric.

832

291

View full text Add to dashboard Cite

Plant sterols are an essential component of the membranes of all eukaryotic organisms. They are either synthesised de novo or taken up from the environment. Their function appears to be to control membrane fluidity and permeability, although some plant sterols have a specific function in signal transduction. The phytosterols are products of the isoprenoid pathway. The dedicated pathway to sterol synthesis in photosynthetic plants occurs at the squalene stage through the activity of squalene synthetase. Although the activity of 3‐hydroxymethyl‐3‐glutaryl coenzyme A (HGMR) is rate‐limiting in the synthesis of cholesterol, this does not appear to be the case with the plant sterols. Up‐regulation of HGMR appears to increase the biosynthesis of cycloartenol but not the Δ5‐sterols. A decline in sterol synthesis is associated with a suppression of squalene synthetase activity, which is probably a critical point in controlling carbon flow and end‐product formation. The major post‐squalene biosynthetic pathway is regulated by critical rate‐limiting steps such as the methylation of cycloartenol into cycloeucalenol. Little is known about the factors controlling the biosynthesis of the end‐point sterol esters or stanols. The commonly consumed plant sterols are sitosterol, stigmasterol and campesterol which are predominantly supplied by vegetable oils. The oils are a rich source of the steryl esters. Less important sources of sterols are cereals, nuts and vegetables. The nutritional interest derives from the fact that the sterols have a similar structure to cholesterol, and have the capacity to lower plasma cholesterol and LDL cholesterol. Since the morbidity and mortality from cardiovascular disease have been dramatically reduced using cholesterol‐lowering drugs (statins), the interest in plant sterols lies in their potential to act as a natural preventive dietary product. Stanols (saturated at C‐5) occur in low amounts in the diet and are equally effective in lowering plasma cholesterol and do not cause an increase in plasma levels, unlike the sterols which can be detected in plasma. © 2000 Society of Chemical Industry

show abstract

Rapid analysis of oxidized cholesterol derivatives by high‐performance liquid chromatography combined with diode‐array ultraviolet and evaporative laser light‐scattering detection

Cited by 21 publications

References 25 publications

Hexavalent Chromium-induced Alteration of Proteomic Landscape in Human Skin Fibroblast Cells

Hexavalent Chromium-induced Alteration of Proteomic Landscape in Human Skin Fibroblast Cells

Quantitative Proteomic Analysis Reveals the Perturbation of Multiple Cellular Pathways in Jurkat-T Cells Induced by Doxorubicin

Plant sterols: biosynthesis, biological function and their importance to human nutrition

Contact Info

Product

Resources

About