External validation of a pharmacokinetic model for target-controlled infusion of cefazolin as a prophylactic antibiotic.

AIMS: This study aimed to evaluate the predictive performance of previously constructed cefazolin pharmacokinetic models and determine whether cefazolin administration via the target-controlled infusion (TCI) method may be possible in clinical practice. METHODS: Twenty-five gastrectomy patients receiving cefazolin as a prophylactic antibiotic were enrolled. Two grams of cefazolin was dissolved in 50 mL of normal saline to give a concentration of 40 mg mL-1 . Before skin incision, cefazolin was administered using a TCI syringe pump, and its administration continued until the end of surgery. The target total plasma concentration was set to 100 µg mL-1 . Total and unbound plasma concentrations of cefazolin were measured in three arterial blood samples collected at 30, 60 and 120 min after the start of cefazolin administration. The predictive performance of the TCI system was evaluated using four measures: inaccuracy, divergence, bias and wobble. RESULTS: Total (n = 75) and unbound (n = 75) plasma concentration measurements from 25 patients were included in the analysis. The pooled median (95% confidence interval) biases and inaccuracies were 6.3 (4.0-8.5) and 10.5 (8.6-12.4) for the total concentration model and -10.3 (-16.8 to -3.7) and 22.4 (18.2-26.7) for the unbound concentration model, respectively. All unbound concentrations were above 10 µg mL-1 . CONCLUSION: Administration of cefazolin by the TCI method showed a clinically acceptable performance. Applying the TCI method by setting the total concentration as the target concentration rather than the unbound concentration is effective in maintaining a constant target concentration of cefazolin.

The hypothalamic-pituitary-gonadal axis controls muscle stem cell senescence through autophagosome clearance.

BACKGROUND: With organismal aging, the hypothalamic-pituitary-gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown. METHODS: We generated muscle stem cell (MuSC)-specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short-term and long-term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30-month-old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed. RESULTS: Disrupting the HPG axis results in impaired muscle regeneration [wild-type (WT) vs. dKO, P < 0.0001; Veh vs. Antide, P = 0.004]. The expression of DNA damage marker (in WT = 7.0 ± 1.6%, dKO = 32.5 ± 2.6%, P < 0.01; in Veh = 13.4 ± 4.5%, Antide = 29.7 ± 5.5%, P = 0.028) and senescence-associated ß-galactosidase activity (in WT = 3.8 ± 1.2%, dKO = 10.3 ± 1.6%, P < 0.01; in Veh = 2.1 ± 0.4%, Antide = 9.6 ± 0.8%, P = 0.005), as well as the expression levels of senescence-associated genes, p16Ink4a and p21Cip1 , was significantly increased in the MuSCs, indicating that genetic and pharmacological inhibition of the HPG axis recapitulates the progressive aging process of MuSCs. Mechanistically, the ablation of sex hormone signalling reduced the expression of transcription factor EB (Tfeb) and Tfeb target gene in MuSCs, suggesting that sex hormones directly induce the expression of Tfeb, a master regulator of the autophagy-lysosome pathway, and consequently autophagosome clearance. Transduction of the Tfeb in naturally aged MuSCs increased muscle mass [control geriatric MuSC transplanted tibialis anterior (TA) muscle = 34.3 ± 2.9 mg, Tfeb-transducing geriatric MuSC transplanted TA muscle = 44.7 ± 6.7 mg, P = 0.015] and regenerating myofibre size [eMyHC+ tdTomato+ myofibre cross-section area (CSA) in control vs. Tfeb, P = 0.002] after muscle injury. CONCLUSIONS: Our data show that the HPG axis systemically controls autophagosome clearance in MuSCs through Tfeb and prevents MuSCs from senescence, suggesting that sustained HPG activity throughout life regulates autophagosome clearance to maintain the quiescence of MuSCs by preventing senescence until advanced age.

Clinical and biochemical relevance of monounsaturated fatty acid metabolism targeting strategy for cancer stem cell elimination in colon cancer.

Lipid metabolism is associated with colon cancer prognosis and incidence. Stearoyl-CoA desaturase 1 (SCD1), which converts fully saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs), has been suggested as a vulnerable target for selective elimination of cancer stem cells (CSCs). However, the clinical significance and physiological role of SCD1 in CSCs has not been well demonstrated. Here, we showed the clinical and biochemical relevance of blocking SCD1 to target CSCs by analyzing human colon cancer data from TCGA and through lipidomic profiling of CSCs with or without SCD1 inhibition using mass spectrometry. Positive associations between SCD1 expression and colorectal cancer patient clinical status and the expression of CSC-related genes (WNT and NOTCH signaling) were found based on TCGA data analysis. Lipidomic profiling of CSCs and bulk cancer cells (BCCs) using mass spectrometry revealed that colon CSCs contained a distinctive lipid profile, with higher free MUFA and lower free SFA levels than in BCCs, suggesting that enhanced SCD1 activity generates MUFAs that may support WNT signaling in CSCs. In addition, all identified phosphatidyl-ethanolamine-containing MUFAs were found at higher levels in CSCs. Interestingly, we observed lower phosphatidyl-serine (18:1/18:0), phosphatidyl-choline (PC; p-18:0/18:1)), and sphingomyelin (SM; d18:1/20:0 or d16:1/22:0) levels in CSCs than in BCCs. Of those, SCD1 inhibition, which efficiently diminished free MUFA levels, increased those specific PC and SM and MUFAs in CSCs promptly. These results suggest that these specific lipid composition is critical for CSC stem cell maintenance. In addition, not only free MUFAs, which are known to be required for WNT signaling, but also other phospholipids, such as SM, which are important for lipid raft formation, may mediate other cell signaling pathways that support CSC maintenance. Comparison of the lipidomic profiles of colon cancer cells with those of previously reported for glioma cells further demonstrated the tissue specific characteristics of lipid metabolism in CSCs.

Increased biosynthesis and accumulation of cholesterol in maternal plasma, but not amniotic fluid in pre-eclampsia.

Preeclampsia is one of the most serious complications during pregnancy, defined as development of hypertension during late pregnancy affecting other organ systems (proteinuria, thrombocytopenia, renal insufficiency, liver involvement, cerebral symptoms or pulmonary edema). Preeclampsia is known to be associated with significant dyslipidemia, but the cause or mechanism of this metabolic aberration is not clear. Quantitative analysis of cholesterol precursors and metabolites can reveal metabolic signatures of cholesterol, and provide insight into cholesterol biosynthetic and degradation pathways. We undertook this study to compare the metabolic signatures of cholesterol in serum and amniotic fluid collected from women who delivered in the late preterm period. Matching serum and amniotic fluid samples were collected from women who delivered in the late preterm period (34-0/7-36-6/7 weeks), had undergone amniocentesis within 3 days of delivery, had no evidence of rupture of membranes or intra-amniotic infection/inflammation, and who had not received antenatal corticosteroid prior to amniocentesis. Patients were classified into 3 groups according to the etiology of their preterm birth: Group 1, preeclampsia; Group 2, spontaneous preterm labor; Group 3, other maternal medical indications for iatrogenic preterm birth. Quantitative metabolite profiling of cholesterols was performed using gas chromatography-mass spectrometry. A total of 39 women were included in the analysis (n = 14 in Group 1, n = 16 in Group 2, n = 9 in Group 3). In maternal blood, patients in Group 1 had significantly higher ratios of cholesterol/desmosterol and cholesterol/7-dehydrocholesterol (which represent 24- and 7-reductase enzyme activity, respectively) than those in Group 3 (p < 0.05 for each), which suggests increased cholesterol biosynthesis. In contrast, patients in Group 1 had significantly decreased ratios of individual cholesterol esters/cholesterol and total cholesterol esters/cholesterol than those in Groups 3 (p < 0.01 for each), suggesting increased reverse cholesterol transport. No differences in cholesterol ratios were found in amniotic fluid among the 3 groups. In conclusion, the metabolic signatures of cholesterol suggest increased cholesterol biosynthesis and accumulation in the maternal blood (but not amniotic fluid) of women with preeclampsia.

Effects of alternate day fasting and exercise on cholesterol metabolism in overweight or obese adults: A pilot randomized controlled trial.

BACKGROUND AND PURPOSE: The objective of this pilot randomized controlled trial was to investigate the effect of alternate day fasting (ADF) and exercise on serum sterol signatures, which are surrogate markers of cholesterol absorption and biosynthesis. METHODS: We randomly assigned 112 overweight or obese participants to four groups: 1) ADF and exercise (E-ADF); 2) ADF; 3) exercise; and 4) control. We studied 31 completers in this exploratory analysis and measured their serum sterol signatures using gas chromatography-mass spectrometry. RESULTS: After intervention, most serum sterol signatures that correspond to cholesterol metabolism were significantly different between groups (p < 0.05 by analysis of covariance [ANCOVA]). We found no differences in plant sterols, which are markers of cholesterol absorption. In the exercise group, desmosterol, cholesteryl esters, and oxysterols decreased significantly. Furthermore, only changes in physical activity levels negatively correlated with changes in the metabolic ratios of desmosterol and 7-dehydrocholesterol to cholesterol, which reflect cholesterol biosynthesis (r = -0.411; p = 0.030, and r = -0.540; p = 0.003, respectively). CONCLUSION: These findings suggest that exercise with or without ADF improves cholesterol metabolism as measured by serum sterol signatures, and increased physical activity has a greater effect on cholesterol biosynthesis than weight reduction or calorie restriction.

Supported liquid extraction coupled to gas chromatography-selective mass spectrometric scan modes for serum steroid profiling.

Although, steroid profiling is being applied in clinical and biochemical studies, improvement of the technique is still needed for accurate quantification of steroids in case of limited biological sample volumes. To improve analytical sensitivity and selectivity in comparison to that of conventional methods, a method that comprises supported liquid extraction (SLE) and gas chromatography-mass spectrometry with a combination of selected-reaction and selected-ion monitoring modes (GC-SRM/SIM-MS) was developed. Here, this combination of SLE purification with GC-MS method was optimized with 37 different types of steroids and the results were compared to a solid-phase extraction (SPE) method. The devised assay led to an increase in extraction efficiency with the good chromatographic selectivity through a single extraction step. The limits of quantification of the serum steroids, ranged from 0.2 to 5 ng mL-1, except for cholesterol (0.2 µg mL-1), and the correlation coefficients for calibration curves were higher than 0.99. The precision and accuracy were 1.4%-10.5% and 82.7%-115.3%, respectively. The overall recoveries of 30 steroids ranged from 62.1% to 104.3%, while that of 7 sterols was 44.7%-75.7%. Then, this validated method was applied to monitor the serum steroid levels of mice, which showed significant sex and age dependent metabolic patterns. This technique can be used to evaluate the metabolic changes occurring in animal models as well as in clinical patients.

Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids.

Housekeeping metabolic pathways such as glycolysis are active in all cell types. In addition, many types of cells are equipped with cell-specific metabolic pathways. To properly perform their functions, housekeeping and cell-specific metabolic pathways must function cooperatively. However, the regulatory mechanisms that couple metabolic pathways remain largely unknown. Recently, we showed that the steroidogenic cell-specific nuclear receptor Ad4BP/SF-1, which regulates steroidogenic genes, also regulates housekeeping glycolytic genes. Here, we identify cholesterogenic genes as the targets of Ad4BP/SF-1. Further, we reveal that Ad4BP/SF-1 regulates Hummr, a candidate mediator of cholesterol transport from endoplasmic reticula to mitochondria. Given that cholesterol is the starting material for steroidogenesis and is synthesized from acetyl-CoA, which partly originates from glucose, our results suggest that multiple biological processes involved in synthesizing steroid hormones are governed by Ad4BP/SF-1. To our knowledge, this study provides the first example where housekeeping and cell-specific metabolism are coordinated at the transcriptional level.

Simultaneous quantification of 18 saturated and unsaturated fatty acids and 7 sterols as their tert-butyldimethylsilyl derivatives in human saliva using gas chromatography-tandem mass spectrometry.

The profiling of fatty acids (FAs) or sterols has been applied in clinical studies, but still needs to be improved to enable their simultaneous quantification. Moreover, little progress has been made in determining the levels of FAs and sterols in human saliva in a single run. In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) using one-step tert-butyldimethylsilyl (TBDMS) derivatization was developed for comprehensive profiling of 18 FAs (eight saturated, five monounsaturated, and five polyunsaturated FAs) and 7 sterols (cholesterol and its precursors). The TBDMS derivatization process was also optimized in terms of reaction solvent, catalyst, temperature, and reaction time. The optimized conditions resulted in better derivatization efficiency with good chromatographic separation through a high-temperature column within 23 min. The present method provided good linearity (r > 0.993), precision (coefficient of variation, 2.7% to 10.4%), and accuracy (91.5% to 103.4%). The overall recovery ranged from 73.8% to 114.3% for the 18 FAs, and from 68.9% to 79.8% for the 7 sterols. The validated method was applied to characterize FAs and sterols in human saliva samples. This is the first report of a GC-MS/MS method for the simultaneous determination of various FAs and sterols from a small volume (100 µL) of saliva. This approach can be used as a primary screening tool to examine the levels of both FAs and sterols in saliva, providing detailed information about their homeostasis for diagnostic and prognostic purposes.

Targeted and non-targeted metabolite identification of MAM-2201 in human, mouse, and rat hepatocytes.

MAM-2201 is a fluorinated naphthoylindole synthetic cannabinoid with potent psychoactive properties that has been detected as an active ingredient in herbal incense blends. To gain a greater understanding of MAM-2201 metabolism and to compare its metabolic fate in humans with those in animals, the metabolism of MAM-2201 in human, mouse, and rat hepatocytes was investigated using liquid chromatography-high-resolution mass spectrometry combined with targeted and non-targeted metabolite profiling approaches. Nineteen phase I metabolites (M1-M19) reported previously in human liver microsomes and 13 novel metabolites were identified in human, mouse, and rat hepatocytes: 1 phase I metabolite (M20) and 12 phase II metabolites including 6 glucuronides (G1-G6), 1 sulfate (S1), and 5 glutathione (GSH) conjugates (GS1-GS5) of MAM-2201 metabolites. G3 was human-specific, but M20, G1, G2, and 5 GSH conjugates were rat-specific, indicating species-related differences in MAM-2201 metabolism. The findings in the present study can be useful for the experimental design and assessment of metabolism-mediated toxic risk of MAM-2201.

Metabolomics-assisted metabolite profiling of itraconazole in human liver preparations.

Itraconazole (ITZ) is a first-generation triazole-containing antifungal agent that effectively treats various fungal infections. As ITZ has a better safety profile than that of ketoconazole (KCZ), ITZ has been used worldwide for over 25 years. However, few reports have explored the metabolic profile of ITZ, and the underlying mechanism of ITZ-induced liver injury is not clearly understood. In the present study, we revisited ITZ metabolism in humans, using a non-targeted metabolomics approach, and identified several novel metabolic pathways including O-dearylation, piperazine oxidation, and piperazine-N,N'-deethylation. Furthermore, we explored the formation of reactive ITZ metabolites using trapping agents as surrogates, to assess the possibility of metabolism-mediated toxicity. We found that ITZ and its metabolites did not form any adducts with nucleophiles including glutathione, potassium cyanide, and semicarbazide. The present study expands our knowledge of ITZ metabolism and supports the suggestion that ITZ has a better safety profile than that of KCZ in terms of metabolism-mediated toxicity.

Improved detectability of sex steroids from frozen sections of breast cancer tissue using GC-triple quadrupole-MS.

Sex steroids in clinical endocrinology have been mainly investigated with peripheral blood and urine samples, while there is limited information regarding the local levels within tissues. To improve analytical properties of sex steroids from trace amounts of tissue samples, two-phase extractive ethoxycarbonlyation and subsequent pentafluoropropionyl derivatization coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed. The optimized analytical conditions led to excellent chromatographic separation of 15 estrogens, 6 androgens, and 2 progestins. The quantitative results were calculated based on in-house control samples as the steroid-free tissues, and the precision and accuracy were 4.2%-26.8% and 90.8%-116.4%, respectively. The on-column limit of quantification was from 180 fg to 0.5 pg for androgens and estrogens, and 1.25 pg for progestins, which were found to be linear (r2 > 0.990). The validated method was then applied to quantify 7 sex steroids from three 100-µm-thick frozen breast tissue slices from postmenopausal patients with breast cancer. This is the first report on the improved GC-MS/MS method for the detection of androgens and pregnenolone from breast cancer tissues, and it can be a useful technique to measure the local levels of sex steroids, thus, enhancing our understanding of the pathophysiological significances of steroidogenesis.

Effects of cytokines derived from cancer-associated fibroblasts on androgen synthetic enzymes in estrogen receptor-negative breast carcinoma.

PURPOSE: The tumor microenvironment plays pivotal roles in promotion of many malignancies. Cancer-associated fibroblasts (CAFs) have been well-known to promote proliferation, angiogenesis, and metastasis but mechanistic understanding of tumor-stroma interactions is not yet complete. Recently, estrogen synthetic enzymes were reported to be upregulated by co-culture with stromal cells in ER positive breast carcinoma (BC) but effects of co-culture on androgen metabolism have not been extensively examined. Therefore, we evaluated roles of CAFs on androgen metabolism in ER-negative AR-positive BC through co-culture with CAFs. METHODS: Concentrations of steroid hormone in supernatant of co-culture of MDA-MB-453 and primary CAFs were measured using GC-MS. Cytokines derived from CAFs were determined using Cytokine Array. Expressions of androgen synthetic enzymes were confirmed using RT-PCR and Western blotting. Correlations between CAFs and androgen synthetic enzymes were analyzed using triple-negative BC (TNBC) patient tissues by immunohistochemistry. RESULTS: CAFs were demonstrated to increase expressions and activities of 17ßHSD2, 17ßHSD5, and 5α-Reductase1. IL-6 and HGF that were selected as potential paracrine mediators using cytokine array induced 17ßHSD2, 17ßHSD5, and 5α-Reductase1 expression. Underlying mechanisms of IL-6 paracrine regulation of 17ßHSD2 and 17ßHSD5 could be partially dependent on phosphorylated STAT3, while phosphorylated ERK could be involved in HGF-mediated 5α-Reductase1 induction. α-SMA status was also demonstrated to be significantly correlated with 17ßHSD2 and 17ßHSD5 status in TNBC tissues, especially AR-positive cases. CONCLUSIONS: Results of our present study suggest that both IL-6 and HGF derived from CAFs could contribute to the intratumoral androgen metabolism in ER-negative BC patients.

Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations.

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

Body Fat Mass Is Associated With Ratio of Steroid Metabolites Reflecting 17,20-Lyase Activity in Prepubertal Girls.

CONTEXT: Pediatric obesity has been related to hyperandrogenism and premature adrenarche in previous studies. However, little is known regarding the association between body fat mass and steroidogenic enzyme activities in children. OBJECTIVE: To examine whether body fat mass is associated with serum steroid profiles in girls. DESIGN, PARTICIPANTS, AND SETTING: We enrolled 242 girls (125 prepubertal, 117 pubertal; age, 7-13 years). Early morning blood samples were drawn at a university hospital to measure serum steroid profiles using gas chromatography-mass spectrometry, and steroidogenic enzyme activities were assessed from the ratios of steroid metabolites. MAIN OUTCOME MEASURES: We evaluated serum steroid profiles and estimated steroidogenic enzyme activities and their association with anthropometric indices and body composition. RESULTS: Prepubertal obese girls demonstrated significantly higher progestin, androgens (dehydroepiandrosterone [DHEA], androstenedione [A-dione], T, androsterone), and ratio of steroid metabolites reflecting 17,20-lyase activity [(DHEA + A-dione)/17-hydroxypregnenolone] compared with prepubertal controls. Pubertal obese girls demonstrated significantly higher serum T and androsterone than pubertal controls; however, serum steroid metabolite ratios reflecting steroidogenic enzyme activities did not significantly differ among obese and non-obese girls. Partial correlation analysis revealed that body fat mass was positively correlated with pregnenolone, DHEA, A-dione, T, androsterone, and ratio of (DHEA + A-dione)/17-hydroxypregnenolone in prepubertal girls only. Prepubertal girls with increased body fat mass had significantly higher serum DHEA and ratio of (DHEA + A-dione)/17-hydroxypregnenolone than controls. CONCLUSIONS: Increased androgen production in prepubertal obese girls could be at least partly due to increased body fat mass and 17,20-lyase activity.

Urinary 6ß-Hydroxycortisol/Cortisol Ratio Most Highly Correlates With Midazolam Clearance Under Hepatic CYP3A Inhibition and Induction in Females: A Pharmacometabolomics Approach.

Endogenous metabolites of cytochrome P450 (CYP3A) are useful in predicting drug-drug interactions between in vivo CYP3A inhibitors and inducers for clinical applications of CYP3A substrate drugs. This study aimed to develop predictable markers of the magnitude of hepatic CYP3A induction and inhibition in healthy female subjects using pharmacometabolomics. Twelve female subjects received midazolam during three study phases: 1 mg midazolam (control phase), 1 mg midazolam after pretreatment with 400 mg ketoconazole once daily for 4 days (CYP3A inhibition phase), and 2.5 mg midazolam after pretreatment with 600 mg rifampicin once daily for 10 days (CYP3A induction phase). Throughout the study, blood samples were collected 24 h after midazolam administration and urine samples at 12-h intervals during the 24 h before and after midazolam administration for the analysis of endogenous steroid metabolites. A statistical model was generated to predict midazolam clearance using measurements of endogenous metabolites associated with the inhibition and induction of CYP3A. Mean midazolam clearance decreased to ∼20% of control levels during the inhibition phase and increased more than 2-fold during the induction phase. Of the urine and plasma metabolites measured, the 6ß-hydroxycortisol/cortisol ratio was most significantly correlated with midazolam clearance during hepatic CYP3A inhibition and induction. Our results suggest that the urinary 6ß-hydroxycortisol/cortisol ratio is the best predictor of hepatic CYP3A activity under both maximal inhibition and maximal induction. Furthermore, the predictive model including 6ß-hydroxycortisol/cortisol as a covariate could be applied to predict the magnitude of CYP3A-mediated drug interactions.

Hair sterol signatures coupled to multivariate data analysis reveal an increased 7ß-hydroxycholesterol production in cognitive impairment.

Altered cholesterol metabolism could be associated with cognitive impairment. The quantitative profiling of 19 hair sterols was developed using gas chromatography-mass spectrometry coupled to multivariate data analysis. The limit of quantification of all sterols ranged from 5 to 20 ng/g, while the calibration linearity was higher than 0.98. The precision (% CV) and accuracy (% bias) ranged from 3.2% to 9.8% and from 83.2% to 119.4%, respectively. Among the sterols examined, 8 were quantitatively detected from two strands of 3-cm-long scalp hair samples of female participants, including mild cognitive impairment (MCI, n=15), Alzheimer's disease (AD, n=31), and healthy controls (HC, n=36). The cognitive impairment (MCI or AD) was correlated with a higher metabolic rate than that of HCs based on 7ß-hydroxycholesterol (P<0.005). Significant negative correlations (r=-0.822) were detected between Mini-Mental State Examination (MMSE) scores and hair sample metabolic ratios of 7ß-hydroxycholesterol to cholesterol, which is an accepted, sensitive, and specific tool for discriminating HCs from individuals with MCI or AD. In conclusion, improved diagnostic values can be obtained using hair sterol signatures coupled with MMSE scores. This method may prove useful for predictive diagnosis in population screening of cognitive impairment.

Serum sterol profiling reveals increased cholesterol biosynthesis in childhood obesity.

Quantitative sterol profiling in obese children and their clinical implications have not been fully investigated. The aim of study was to evaluate the metabolic changes in serum cholesterol and its precursors and metabolites, and their associations with clinical characteristics of childhood obesity. A total of 253 children aged 6-14 years (72 obese, 39 overweight, and 72 normal controls; 147 girls and 106 boys) were recruited. Anthropometric indices, body composition, and fasting total lipid profiles were determined. Serum concentrations of 20 sterols, as their free fraction, were analyzed through gas chromatography-mass spectrometry-based metabolite profiling. There were no significant differences in total- and LDL-cholesterols between groups. Serum levels of the main cholesterol precursors, lanosterol (P<0.02) and lathosterol (P<0.0001), were significantly higher in obese children. In addition, they showed positive correlations with waist to hip ratio, body fat percent, and body fat mass. The metabolic ratios of lanosterol and lathosterol to cholesterol were also elevated (P<0.01 both), indicating the up-regulation of cholesterol biosynthesis with childhood obesity. In contrast, the absorption of plant sterols tended to show a compensatory decrease in obese children. Strong correlations between free cholesterol and total- and LDL-cholesterols were observed (r>0.760, P<0.001), while there was no correlation with HDL-cholesterols. The levels of total cholesteryl ester were closely associated with triglyceride (r=0.763, P<0.001). Quantitative results indicate that childhood obesity may increase cholesterol synthesis while maintaining overall cholesterol homeostasis.

Metabolic changes in serum steroids induced by total-body irradiation of female C57B/6 mice.

The short- and long-term effects of a single exposure to gamma radiation on steroid metabolism were investigated in mice. Gas chromatography-mass spectrometry was used to generate quantitative profiles of serum steroid levels in mice that had undergone total-body irradiation (TBI) at doses of 0Gy, 1Gy, and 4Gy. Following TBI, serum samples were collected at the pre-dose time point and 1, 3, 6, and 9 months after TBI. Serum levels of progestins, progesterone, 5ß-DHP, 5α-DHP, and 20α-DHP showed a significant down-regulation following short-term exposure to 4Gy, with the exception of 20α-DHP, which was significantly decreased at each of the time points measured. The corticosteroids 5α-THDOC and 5α-DHB were significantly elevated at each of the time points measured after exposure to either 1 or 4Gy. Among the sterols, 24S-OH-cholestoerol showed a dose-related elevation after irradiation that reached significance in the high dose group at the 6- and 9-month time points.

Cytochrome P450-mediated metabolic alterations in preeclampsia evaluated by quantitative steroid signatures.

Although preeclampsia has been suggested potential risk factors including placental and systemic inflammation, oxidative stress, and abnormal steroid metabolism during pregnancy, the pathogenesis of preeclampsia has not fully been elucidated, particularly in steroid metabolism. The association between various cytochrome P450 (CYP)-mediated steroid metabolic markers and preeclampsia risk was therefore investigated. The serum levels of 54 CYP-mediated regioselective hydroxysteroids and their substrates were quantitatively evaluated from both pregnant women with preeclampsia (n=30; age, 30.8±4.5 years) and normotensive controls (n=30; age, 31.0±3.5 years), who were similar with respect to maternal age, gestational age, and body mass index. The levels of 6ß-, 7a-, and 11ß-hydroxymetabolites of androgens and corticoids were significantly increased in women with preeclampsia. In addition, the levels of oxysterols, including 7a-, 7ß-, 4ß-, 20a-, 24S-, and 27-hydroxycholesterol, were markedly higher, while the levels of 16a-OH-DHEA, 16a-OH-androstenedione, and cholesterol were significantly decreased in patients. The 6ß-hydroxylation of androgens and corticoids by CYP3A4 (P<0.01), the activation of 20,22-desmolase (a cholesterol side-chain cleavage enzyme) by CYP11A1 (P<0.00001), and the multi-hydroxylation of cholesterol at C-4ß, C-7a, C-7ß, C-24S, C-27, and C-20a (P<0.0001) by catalytic or enzymatic reaction (e.g. CYP3A4, CYP7A1, CYP27A1, and CYP46A1) were differed between preeclamptic women and control subjects. In particular, an increased oxysterols (induction>2.0-fold) were positively correlated with the conditions of preeclampsia. Our metabolic profiling suggests the CYP-mediated alterations in steroid metabolism and hydroxylation in pregnancy-induced hypertension. These multiple markers could serve as background information for improved clinical diagnosis and management during pregnancy. This article is part of a Special Issue entitled "Pregnancy and Steroids".

High-temperature GC-MS-based serum cholesterol signatures may reveal sex differences in vasospastic angina.

Alterations of cholesterol metabolism are responsible for vasospastic angina and atherosclerosis. To comprehensively evaluate cholesterol metabolism, 18 sterols, including cholesterol, 6 cholesteryl esters (CEs), 3 cholesterol precursors, and 8 hydroxycholesterols (OHCs), were simultaneously analyzed using hybrid solid-phase extraction (SPE) purification coupled to high-temperature gas chromatography-mass spectrometry (HTGC-MS). Methanol-based hybrid SPE increased the selective extraction, and HTGC resulted in a good chromatographic resolution for the separation of lipophilic compounds. The limits of quantification of cholesterol and CEs ranged from 0.2 to 10.0 µg/ml, while OHCs and cholesterol precursors ranged from 0.01 to 0.10 µg/ml. Linearity as the correlation coefficient was higher than 0.99 with the exception of cholesteryl laurate, myristate, oleate, and linoleate (r² > 0.98). The precision (% coefficient of variation) and accuracy (% bias) ranged from 1.1 to 9.8% and from 75.9 to 125.1%, respectively. The overall recoveries of CEs ranged from 26.1 to 64.0%, and the recoveries of other sterols ranged from 83.8 to 129.3%. The cholesterol signatures showed sex differences in patients with vasospastic angina and may associate with 24-reductases. This technique can be useful for making clinical diagnoses and for an increased understanding of the pathophysiology of vasospastic angina.