Quantitative Determination of Cholesterol Hydroxylase Specificities by GC-MS/MS in Living Mammalian Cells.

Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors. Each oxysterol has distinct and overlapping functions. The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood. Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively. To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required. Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases-CH25H, CYP7A1, CYP27A1, and CYP46A1-or induction of CH25H expression by a physiological stimulus. This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity. Key features • Allows measuring the amount of a variety of oxysterols synthesized endogenously by cholesterol hydroxylases using GC-MS/MS. • Comprehensive and quantitative analysis of cholesterol hydroxylase specificities in living mammalian cells. • Simultaneous quantification of intermediate sterols to assess cholesterol synthesis activity.

Hydroxylation site-specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR.

The cholesterol metabolites, oxysterols, play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

Usefulness of criteria for intraoperative Management of Postoperative Nausea and Vomiting.

BACKGROUND: Postoperative nausea and vomiting (PONV) delays postoperative recovery, prolongs hospital stays, and hinders patients' return to society, thus making it a major cause of increased healthcare costs. It is also the most troubling postoperative complication in female patients undergoing surgery. However, in Japan, guidelines for the management of PONV have not been established, and the management protocol for PONV is left to each institution and anesthesiologist. Therefore, we developed criteria for intraoperative management of PONV. METHODS: In female surgical patients, the usefulness of the criteria was evaluated by comparing the implementation rate of intraoperative management and PONV incidence before and after the establishment of the criteria. An Apfel simplified score (Apfel score) ≥2 was set as an indication for intraoperative management of PONV. RESULTS: The implementation rate of intraoperative management increased from 91.2 to 96.0% after the introduction of the criteria. In patients with an Apfel score of 2, the intraoperative management implementation rate significantly increased from 81.1 to 94.7% (p = 0.016), while PONV incidence significantly decreased from 44.6 to 34.1% after the introduction of the criteria (p = 0.040). CONCLUSIONS: The criteria for intraoperative management of PONV increased the implementation rate of intraoperative management and decreased PONV incidence, indicating the usefulness of the criteria.

Improvement of a compressed inferior vena cava due to IgG4-related retroperitoneal fibrosis with steroid therapy.

A 79-year-old man had a 3.5-year history of edema of the lower extremities of unknown etiology. Abdominal computed tomography showed a soft tissue mass around the abdominal aorta, and the biopsy revealed dense fibrosis with abundant infiltration of IgG4-positive plasma cells. His serum IgG4 level was increased to 188 mg/dL. His lower extremity edema was induced by stenosis of the inferior vena cava (IVC) due to the mass. With a diagnosis of IgG4-related retroperitoneal fibrosis, he was treated with steroid, and the leg edema decreased with improvement of patency of the IVC and reduction of the soft tissue mass.