Methylprednisolone increases plasma leptin levels in Graves' hyperthyroidism patients with active Graves' ophthalmopathy.

Whether leptin, a product of the ob gene, can be stimulated by glucocorticoid administration has been an issue of controversy. We investigated the effect of intravenous administration of methylprednisolone (500 mg/day x 3 days) on plasma levels of leptin in 16 patients (female/male = 11/5) with Graves' hyperthyroidism and active ophthalmopathy who received pulse therapy. Significant elevation of plasma leptin levels started at the eighth hour (13.9+/-1.8 ng/mL, p=0.042) and lasted until the 72nd hour (21.2+/-5.0 ng/mL, p=0.009), as compared with basal levels (8.8+/-1.2 ng/mL). When methylprednisolone was replaced with oral prednisolone (10 mg three times per day x 2 weeks), no difference in plasma leptin levels was noted compared with basal measurement. Under methylprednisolone administration, a significant suppression of tumor necrosis factor-alpha began at the 24th hour (8.1+/-1.3 pg/mL, p=0.004) and lasted until the 48th hour (8.1+/-1.0 pg/mL, p=0.008), as compared with basal measurement (12.5+/-1.5 pg/mL). Compared with basal levels (93+/-2 mg/dL), significant elevation in the plasma glucose level started at the third hour (135+/-10 mg/dL, p=0.000) and lasted until the 72nd hour (110+/-4 mg/dL, p=0.019). The timing of serum insulin elevation approximated that of plasma glucose (3 hours: 14+/-3 microU/mL, p=0.006) and lasted until the end of prednisolone administration (2 weeks: 12+/-2 microU/mL, p=0.044), when compared with basal levels (14+/-3 microU/mL). We concluded that the parental administration of pharmacological doses of methylprednisolone to patients with Graves' hyperthyroidism could acutely raise their plasma level of leptin.     

Occurrence of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid as normal constituents in human blood

Three unconjugated C27 bile acids were found in plasma from healthy humans. They were isolated by liquid-solid extraction and anion-exchange chromatography and were identified by gas-liquid chromatography-mass spectrometry, microchemical reactions, and ultraviolet spectroscopy as 3 beta-hydroxy-5-cholestenoic, 3 beta,7 alpha-dihydroxy-5-cholestenoic, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acids. Their levels often exceeded those of the unconjugated C24 bile acids and the variations between individuals were smaller than for the C24 acids. The concentrations in plasma from 11 healthy subjects were 67.2 +/- 27.9 ng/ml (mean +/- SD) for 3 beta-hydroxy-5-cholestenoic acid, 38.9 +/- 25.6 ng/ml for 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 81.7 +/- 27.9 ng/ml for 7 alpha-hydroxy-3-oxo-4-cholestenoic acid. The levels of the individual acids were positively correlated to each other and not to the levels of the C24 acids. The cholestenoic acids were below the detection limit (20-50 ng/ml) in bile and C27 bile acids present in bile were not detected in plasma.     

Concentrations of cholestenoic acids in plasma from patients with liver disease

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients with different liver diseases and compared with those of unconjugated and conjugated C24 bile acids. The levels of the cholestenoic acids were similar in patients with extrahepatic cholestasis and in controls (median concentration 153 and 162 ng/ml, respectively), whereas significantly elevated levels were found in plasma from patients with primary biliary cirrhosis (median concentration 298 ng/ml) and alcoholic liver cirrhosis (median concentration 262 ng/ml). As expected, conjugated C24 bile acids were elevated in most patients whereas the corresponding unconjugated compounds were low in cholestasis and elevated in alcoholic liver cirrhosis. The levels of the individual C27 acids were usually positively correlated to each other and also to the levels of conjugated C24 bile acids in plasma from patients with liver cirrhosis. In contrast, there was no correlation between the levels of C27 acids and conjugated bile acids in patients with extrahepatic cholestasis. The levels of unconjugated C24 bile acids were not correlated to C27 acids or conjugated bile acids in any of the groups. The results indicate that there is a close metabolic relationship between the individual C27 acids, that they do not participate in an enterohepatic circulation, and that the liver is important for their elimination/metabolism.     

Plasma levels of ursodeoxycholic acid in black bears, Ursus americanus: seasonal changes

To date, no other studies have examined the seasonal changes in circulating levels of various bile acids in the plasma of wild North American black bears, Ursus americanus. Using gas chromatography, bile acid concentrations were measured in plasma samples obtained during either early or late hibernation, and during summer active periods. Thus, specific compositional changes from individual animals were examined through a given year. Total bile acid concentrations in the plasma of these normal animals were found to range between 0.2 and 3.1 micromol/L (0.9 +/- 0.2 micromol/L, mean +/- SEM). Cholic, ursodeoxycholic and chenodeoxycholic acids were the major bile acid species identified. Ursodeoxycholic acid represented 28.0 +/- 2.6% of the total bile acid pool. Deoxycholic and lithocholic acids were found only in small amounts. In addition, total bile acid concentrations were lower in plasma samples obtained during hibernation compared with those obtained during summer active periods (0.6 +/- 0.1 and 1.2 +/- 0.4 micromol/L, respectively; p < 0.05). However, the relative proportion of ursodeoxycholic acid, was significantly greater in winter than in summer (31.5 +/- 3.2% and 22.2 +/- 4.5%, p < 0.05). Finally, taurine-conjugated bile acids were the predominant species in bear plasma, accounting for >67% of the total bile acids. These data demonstrate that ursodeoxycholic acid is a major bile acid in black bear plasma, mostly conjugated with taurine. Further, the finding of seasonal variation in plasma bile acid composition provides evidence to support the possible role that ursodeoxycholic acid may play in cellular protection in hibernating black bears.     

High-fat diet–induced obesity and insulin resistance were ameliorated via enhanced fecal bile acid excretion in tumor necrosis factor-alpha receptor knockout mice

Tumor necrosis factor-α (TNF-α) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-α receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-α has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-α in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7±3.1 vs. 98.6±3.1 mg/dL, P<0.005), glucose (221.9±14.7 vs. 167.3±8.1 mg/dL, P<0.01), and insulin (5.1±0.3 vs. 3.4±0.3 ng/mL, P<0.05). Fecal excretion of lipid contents was significantly increased in R2KO mice. In R2KO/HFD mice, the decrease in hepatic cholesterol-7a-hydroxylase activity, the rate-limiting enzyme in bile acid synthesis, was inhibited (1.7±0.2 vs. 8.1±1.0 pmol/min/mg protein, P<0.01). These results suggested that HFD-induced obesity with metabolic derangements could be ameliorated in mice lacking TNF-α receptor 2 via increasing fecal bile acid and lipid content excretion. Therefore, TNF-α signaling through TNFR2 is essentially involved in the bile acid synthesis and excretion of lipids, resulting in its beneficial effects.     

Rapid non-enzymatic HPLC determination of total MHPG in human plasma

We have previously reported a method for the determination of total 3-methoxy-4-hydroxy phenylethylene glycol (MHPG) in brain, based on a simple acid-catalyzed hydrolysis. Now we extend this procedure to the determination of plasma total MHPG. The method involves the deproteinization of plasma with perchloric acid, followed by 3 minutes of an acid-catalyzed step. The hydrolysates are injected into the HPLC system, using a formic acid/methanol eluent with fluorimetric detection. Sample detection limit is below 1 ng MHPG/mL of plasma. This procedure has been used for the determination of plasma total MHPG from 109 healthy individuals of both sexes. Mean value was: 5.4 + 2.3 ng total MHPG/mL of plasma (means +/- S.D., N = 109). No sex differences were observed, and a slight correlation with age (r = 0.24, p less than 0.02) has been found. Plasma-free MHPG was also determined in a subgroup of 15 randomly chosen individuals (3.0 +/- 1.2 ng free MHPG/mL plasma, means +/- S.D.). A significant correlation was obtained with plasma total MHPG (r = 0.77, p less than 0.001, N = 15). The main advantage of the present method lays in its simplicity, since no enzymatic hydrolysis or extraction procedures are needed, being its reliability fully proven through 109 plasma total MHPG determinations.     

Homocysteine levels in acromegaly patients

Acromegaly is associated with a two to three-fold increase in mortality related predominantly to cardiovascular disease. The excess mortality is associated most closely with higher levels of growth hormone (GH). Survival in acromegaly may be normalized to a control age-matched rate by controlling GH levels; in particular, GH levels less than 2.5 ng/mL are associated with survival rates equal to those of the general population. Hyperhomocysteinemia has also been recognized as a risk factor for cardiovascular disease, yet there are limited data on the prevalence of hyperhomocysteinemia in patients with acromegaly. Eighteen acromegaly patients (7 male, 11 female, mean age 42.8 +/- 11.0 years) in our endocrine clinic consented to having the following tests performed: complete blood count (CBC), thyroid hormones, folic acid, vitamin B12, plasma homocysteine levels, uric acid, fibrinogen, CRP, fasting glucose, insulin, C-peptide, total serum cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, GH, insulin-like growth factor-1 (IGF-1) and GH levels after an oral glucose tolerance test (OGTT). By history, fourteen had macroadenomas and four had microadenomas; eight had hypertension; two had glucose intolerance, and four had diabetes. Fifteen had had transsphenoidal or transfrontal surgery: two had been cured, but 13 others were taking long-acting octreotide. Five patients had undergone radiotherapy and the acromegaly in two was treated primarily with long-acting octreotide. CBC, thyroid hormone, folic acid, and vit B12 levels were normal in all patients. We divided the patients into two groups according to mean GH levels after an OGTT: Group 1 (GH<2.5 ng/mL, n=10), and Group 2 (GH<2.5 ng/mL, n=8). Comparison of the two groups using Mann-Whitney U testing revealed statistically significant lower levels in Group 1 of the following parameters: GH (1.91 +/- 0.90 vs. 8.58 +/- 5.55 ng/mL, p=0.002), IGF-1 (338.30 +/- 217.90 vs. 509.60 +/- 293.58 ng/dL, p=0.06), GH after an OGTT (1.42 +/- 0.81 vs. 9.01 +/- 4.53 ng/mL, p=0.001), plasma homocysteine (12.85 +/- 4.47 vs. 18.20 +/- 4.99 micromol/L, p=0.05), total cholesterol (164.0 +/- 20.81 vs. 188.0 +/- 22.26 mg/dL, p=0.05) and LDL cholesterol (81.0 +/- 9.64 vs. 116.70 +/- 13.03 mg/dl, p=0.01). Differences between the other parameters were not significantly different. Acromegaly patients with high GH levels after an OGTT have much higher levels of homocysteine than patients with lower GH levels. The role of elevated homocysteine levels as an independent cardiovascular risk factor in the mortality of acromegaly patients should be determined in future studies.     

Simultaneous determination of catechin, epicatechin and epicatechin gallate in rat plasma by LC-ESI-MS/MS for pharmacokinetic studies after oral administration of Cynomorium songaricum extract

A rapid and valid method was developed for simultaneous determination catechin, epicatechin and epicatechin gallate in rat plasmas using scopoletin (103 ng mL(-1)) as an internal standard (IS). The separation was performed on Eclipse plus C18 column (100 mm × 4.6 mm, 1.8 μm) at a flow rate of 0.3 mL min(-1), and acetonitrile-0.1% formic acid was used as mobile phase. The recoveries of three analytes and IS were more than 78.9%. The lower limits of quantitation (LLOQ) in rat plasma were 2.14, 2.38 and 2.08 ng mL(-1) respectively for catechin, epicatechin and epicatechin gallate. Intra-day and inter-day precisions were within 12%. The accuracies were more than 85%. After single oral administration of 15.25 g kg(-1) Cynomorium songaricum extract, C(max) of catechin, epicatechin and epicatechin gallate in rat plasma were respectively 86.69±38.65, 32.57±15.00 and 36.93±12.62 ng mL(-1) while T(max) values were respectively 0.15±0.09, 0.20±0.10 and 0.20±0.13 h. The results demonstrated that the present LC-MS/MS method was sensitive enough for pharmacokinetic study of catichins following oral administration of C. songaricum extract.     

Bile acids in the fetal rat: Effect of maternal bile duct ligation

The effect of bile duct ligation during pregnancy in rats (thereby increasing maternal plasma bile acids levels) on the bile acid content and composition in the fetus was examined. In spite of 30-fold increase in maternal plasma cholic acid, the bile acid content in the fetus of bile duct ligated rats was significantly lower (P <0.05) with a significant reduction in cholic acid content. Plasma cholesterol levels of fetuses from bile duct ligated rats were also significantly lower (p <0.05). In addition to the commonly expected bile acids, gas-liquid Chromatographic analysis of the fetal bile acid pool showed peaks corresponding to several secondary bile acids. These results suggest that the transfer of primary bile acids of maternal origin into the fetus is minimal.     

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究(英文)

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取,汉邦C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇∶水(85∶15,v/v),水相含10 mmol/L的醋酸铵和0.1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测,草乌甲素及内标的反应离子对分别为644.4/584.4和237.2/194.2,草乌甲素血药浓度在0.010～1.0 ng/mL范围内线性关系良好,最低定量限为0.010 ng/mL可以满足口服0.4 mg草乌甲素后血药浓度的检测,日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中,20名志愿者口服0.4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0.325±0.110),(0.323±0.115)ng/mL;AUC0-16(1.627±0.489),(1.732±0.556)ng.h/mL;AUC0-∞(1.730±0.498),(1.831±0.562)ng.h/mL;t1/2(4.26±0.95),(3.80±0.90)h;Tmax(1.34±0.54),(1.83±0.99)h。     

Contraceptive steroids alter the steady-state kinetics of bile acids

Contraceptive steroids increase the ratio of cholic acid to chenodeoxycholic acid in bile. This alteration may contribute to the development of cholesterol gallstones. The objective of this study was to measure the effects of contraceptive steroids on bile acid kinetics and to relate them to changes in cholesterol metabolism. Steady-state kinetics of bile acids were measured in 15 healthy women, on and off contraceptive steroids. Cholic acid synthesis increased 30.3% (P less than 0.025) and its pool increased by 37.4% (P less than 0.025). Chenodeoxycholic acid synthesis decreased 6.4% (P = 0.08) and its pool decreased by 11.8% (P less than 0.05) during use of contraceptive steroids. The fractional turnover rates of both primary bile acids did not change. The changes in kinetics of the primary bile acids were related to alterations in biliary lipid and cholesterol metabolism, separately reported. (J. Lipid Res. 1987. 28: 828-839). During use of contraceptive steroids, total bile acid pool and total bile acid synthesis correlated directly with cholesterol synthesis, assayed in mononuclear leukocytes (r = 0.50 and r = 0.54, respectively) but not with the plasma clearance of chylomicron remnants, measured with retinyl palmitate. The data indicate that contraceptive steroids directly alter the hepatic synthesis of bile acids and suggest that newly synthesized cholesterol may be a preferred substrate for bile acid synthesis during use of contraceptive steroids.     

Fibroblast Growth Factor Receptor 4 (FGFR4) Deficiency Improves Insulin Resistance and Glucose Metabolism under Diet-induced Obesity Conditions

The role of fibroblast growth factor receptor 4 (FGFR4) in regulating bile acid synthesis has been well defined; however, its reported role on glucose and energy metabolism remains unresolved. Here, we show that FGFR4 deficiency in mice leads to improvement in glucose metabolism, insulin sensitivity, and reduction in body weight under high fat conditions. Mechanism of action studies in FGFR4-deficient mice suggest that the effects are mediated in part by increased plasma levels of adiponectin and the endocrine FGF factors FGF21 and FGF15, the latter of which increase in response to an elevated bile acid pool. Direct actions of increased bile acids on bile acid receptors, and other potential indirect mechanisms, may also contribute to the observed metabolic changes. The results described herein suggest that FGFR4 antagonists alone, or in combination with other agents, could serve as a novel treatment for diabetes.     

Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry

A subgroup of peroxisomal disorders, peroxisome biogenesis defects (PBD), can be differentiated by elevated levels of C(27) bile acids in plasma and bile. Patients with peroxisomal disorders, who lack the ability to chain-shorten the C(27) bile acid intermediates into C(24) bile acids, show elevated levels of C(27) bile acids, notably 3 alpha,7 alpha-dihydroxy-5 beta-cholest-26-oic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. C(27) bile acids are normally estimated against other bile acid standards, by time-consuming gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods, in plasma (minimum of 50 microl). In this article we describe the quantitation of unconjugated di- and trihydroxy C(27) bile acids in 5-microl plasma samples and 3-mm blood spots, using deuterium-labeled internal standards. The synthesis of (2)H(3)-labeled di- and trihydroxycoprostanic acids is described. The sample preparation and analysis by electrospray tandem mass spectrometry (ES-MS/MS) takes less than 1 h and features dimethylaminoethyl ester derivatives. The levels of the di- and trihydroxy bile acids are significantly higher in PBD patients than in age-matched control subjects for both plasma and blood spots collected at birth (some stored for up to 18 years). Excellent correlation is observed between the C(26:0)/C(22:0) very long chain fatty acid (VLCFA) ratio and the levels of trihydroxy C(27) bile acids in plasma from PBD patients.The ES-MS/MS method can be used to rapidly screen for PBD patients in plasma samples with elevated C(26:0)/C(22:0) VLCFA ratios and in archived collections of neonatal blood spots. - Johnson, D. W., H. J. ten Brink, R. C. Schuit, and C. Jakobs. Rapid and quantitative analysis of unconjugated C(27) bile acids in plasma and blood samples by tandem mass spectrometry. J. Lipid Res. 2001. 42: 9;-16.     

The newly identified anorexigenic adipokine nesfatin-1 in hemodialysis patients: Are there associations with food intake, body composition and inflammation?

Nesfatin-1 is a recently identified anorexigenic peptide that has been implicated in appetite regulation, weight loss and/or malnutrition. Anorexia and malnutrition are common features of chronic kidney disease (CKD) that predispose patients to worse outcomes. However, the reasons for the occurrence of anorexia in CKD patients are not fully elucidated. The aim of this study was to investigate the association between nesfatin-1 and protein intake and body composition in patients undergoing hemodialysis (HD). Twenty five HD patients from a private Clinic in Rio de Janeiro, Brazil were studied and compared with 15 healthy subjects that were matched for body mass index (BMI), % body fat mass (by anthropometrics) and age. Appetite was measured using a specific questionnaire, and food intake was evaluated based on 3-day food records. Nesfatin-1 levels were measured by ELISA and leptin, TNF-α and IL-6 levels were determined by a multiplex assay kit. Serum nesfatin-1 levels did not differ between HD patients (0.16±0.07ng/mL) and healthy subjects (0.17±0.10ng/mL). Nesfatin-1 levels showed significant negative correlations with protein intake (r=-0.42; p=0.03), but did not associate with inflammatory markers or appetite scores. Combining patients and controls, we observed positive correlations with BMI (r=0.33; p=0.03), % body fat (r=0.35; p=0.03), leptin (r=0.45; p=0.006) and the triceps skinfold thickness (r=0.36; p=0.02). In multivariate analysis % body fat was the main determinant of nesfatin-1 variance. In conclusion, nesfatin-1 levels did not differ between HD patients and healthy subjects and negatively correlated with protein intake. This pathway is likely not dysregulated in uremia.     

Effects of oestrus induction with progestagens or prostaglandin analogues on ovarian and pituitary function in sheep

The aim of the current study was to determine possible differences in ovarian and pituitary features explaining lower fertility rates in sheep with oestrus induced with intravaginal progestagens or prostaglandin analogues (group FGA and PGF, n=8 in both) when compared to a control group (group C, n=8). The growth profiles and the mean individual sizes of preovulatory follicles were similar between groups; however, the number of preovulatory follicles per ewe and, consequently, the number of ovulations were higher in groups FGA and PGF (2.3±0.3 and 2.0±0.1, respectively) than in group C (1.4±0.1, P<0.05). However, plasma oestradiol concentrations were similar between groups suggesting a defective function in some preovulatory follicles of groups FGA and PGF. In group FGA, the basal LH levels during the follicular phase were lower (0.21±0.0 ng/mL, P<0.005) than in groups C (0.41±0.1 ng/mL) and PGF (0.55±0.1 ng/mL); the onset of preovulatory discharge being later (21.0±2.3h vs. 12.8±1.5 in C and 14.5±1.5 in PGF; P<0.05 for both). Finally, luteal activity was also found to be affected in group FGA; the rate of progesterone secretion per total luteal tissue was lower (range: 0.46-0.65 ng/mL/cm(2)) than in ewes treated with cloprostenol (2.1-3.3 ng/mL/cm(2)) and control sheep (2.0-3.4 ng/mL/cm(2)).     

Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cycling of bile acids in mice

The ileal apical sodium bile acid cotransporter participates in the enterohepatic circulation of bile acids. In patients with primary bile acid malabsorption, mutations in the ileal bile acid transporter gene (Slc10a2) lead to congenital diarrhea, steatorrhea, and reduced plasma cholesterol levels. To elucidate the quantitative role of Slc10a2 in intestinal bile acid absorption, the Slc10a2 gene was disrupted by homologous recombination in mice. Animals heterozygous (Slc10a2+/-) and homozygous (Slc10a2-/-) for this mutation were physically indistinguishable from wild type mice. In the Slc10a2-/- mice, fecal bile acid excretion was elevated 10- to 20-fold and was not further increased by feeding a bile acid binding resin. Despite increased bile acid synthesis, the bile acid pool size was decreased by 80% and selectively enriched in cholic acid in the Slc10a2-/- mice. On a low fat diet, the Slc10a2-/- mice did not have steatorrhea. Fecal neutral sterol excretion was increased only 3-fold, and intestinal cholesterol absorption was reduced only 20%, indicating that the smaller cholic acid-enriched bile acid pool was sufficient to facilitate intestinal lipid absorption. Liver cholesteryl ester content was reduced by 50% in Slc10a2-/- mice, and unexpectedly plasma high density lipoprotein cholesterol levels were slightly elevated. These data indicate that Slc10a2 is essential for efficient intestinal absorption of bile acids and that alternative absorptive mechanisms are unable to compensate for loss of Slc10a2 function.     

Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3-40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, less than 1.5 ng/ml (range less than 0.9-3); liver cirrhosis less than 1.5 ng/ml (range less than 0.9-38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54-477); ileal resection 397 ng/ml (range 128-750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.     

Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: application to a population pharmacokinetics study in children with severe malnutrition

Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm×4.6 mm i.d., 5 μm particle size) maintained at 40°C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02-4 μg/mL, with correlation coefficients (r(2))≥0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8±12.5% (n=5), 83.5±5.2% and 77.7±2.0%, respectively (n=8 in both cases). The recovery for IS was 94.5±7.9% (n=15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at -15°C to -25°C and -70°C to -90°C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.     

A study of the conversion of cholesterol to bile acids by the use of a fistulated bile duct

A method for the surgical preparation of a bile fistula in the sculpin Myoxocephalus quadricornis is presented. This model was used in demonstrating the conversion of ¹⁴C-cholesterol into bile acids. The main acid metabolite was cholic acid and the presence of dihydroxy bile acids was also demonstrated. The amount of labelled compounds which was recovered in the bile was small both in case of parenteral administration to fistulated sculpins and after peroral administration to intact animals. This may be attributable to slow metabolism at the low ambient temperature these animals require to survive.     

Dynamic oral administration of uridine affects the diurnal rhythm of bile acid and cholesterol metabolism-related genes in mice

Bile acids and cholesterol metabolism exhibits distinct daily rhythms and uridine closely associated with bile acids has been well documented. However, how dynamic oral administration of uridine affects bile acid and cholesterol metabolism has not been studied. We conducted the present study to investigate effects of oral administration of uridine in the daytime and nighttime (D-UR and N-UR) on bile acid and cholesterol metabolism-related genes expression in liver and ileum of mice. The results showed that oral administration of uridine in the nighttime (N-UR) reduced serum CHOL and ALT levels at Zeitgeber time (ZT) 4, ZT22, respectively. Compared with D-UR group, the mRNA expression of FXR and SHP genes of liver decreased in N-UR group at ZT10, ZT16, respectively. In addition, oral administration of uridine in the nighttime rhythmically increased the mRNA expression of bile acid transport, cholesterol excretion and decreased the mRNA expression of cholesterol absorption in ileum. Moreover, the expression of nucleotide transport and synthesis genes were also explored in duodenum. Oral administration of uridine in the nighttime rhythmically up-regulated nucleotide transport and synthesis genes expression. In conclusion, these results indicated dynamic oral administration of uridine has effects on the rhythmic fluctuation of cholesterol, bile acid and nucleotide metabolism-related genes. These findings have important physiological and pathophysiological implications, since bile acid and cholesterol metabolism are essential for cell function and closely involved in the development of metabolic syndrome.