Identification of 5-Hydroxy-6-indolyl-O-sulfate in Urine of Patients with Malignant MelanomaIdentification of 5-Hydroxy-6-indolyl-O-sulfate in Urine of Patients with Malignant Melanoma

It has previously been shown that enzymatically hydrolyzed urine of patients with malignant melanoma contains 5,6-dihydroxyindole (5,6DHI). In this study we describe the elucidation of the entire structure of urinary 5,6DHI-conjugate. Differential hydrolysis of melanotic urine revealed that, in contrast to β-glucuronidase, sulfatase can liberate 5,6DHI from its conjugated form. 5,6DHI-sulfate was synthesized by reacting 5,6DHI with sulfur trioxide trimethylamine complex. Thin-layer chromatography (TLC) documented its close similarity to the Thormählen-positive compound usually entitled “C.” Gas chromatographic-mass spectrometric (GC-MS) analysis of methylated and subsequently hydrolyzed synthetic 5,6DHI-sulfate showed that the synthetic product consisted of a mixture of 5-hydroxy-6- indolyl-O-sulfate and 6-hydroxy-5-indolyl-O-sulfate (with a certain amount of 5,6DHI-disulfate). 5,6DHI-sulfate was purified with use of DEAE-cellulose column chromatography from melanotic urine. Methylation of this conjugate with deuterated dimethylsulfate and subsequent GC-MS analysis of the hydrolyzed product provided evidence that 5,6DHI from melanotic urine was almost exclusively sulfated in position 6. It was concluded (1) that 5,6DHI is excreted as a 6-O-sulfate, and (2) that this compound is consistent with Thormählen-positive compound “C.

Colesevelam enhances the beneficial effects of brown fat activation on hyperlipidemia and atherosclerosis development

Aims Brown fat activation accelerates the uptake of cholesterol-enriched remnants by the liver and thereby lowers plasma cholesterol, consequently protecting against atherosclerosis development. Hepatic cholesterol is then converted into bile acids (BAs) that are secreted into the intestine and largely maintained within the enterohepatic circulation. We now aimed to evaluate the effects of prolonged brown fat activation combined with inhibition of intestinal BA reabsorption on plasma cholesterol metabolism and atherosclerosis development and results APOE∗3-Leiden.CETP mice with humanized lipoprotein metabolism were treated for 9 weeks with the selective b3-adrenergic receptor (AR) agonist CL316,243 to substantially activate brown fat. Prolonged b3-AR agonism reduced faecal BA excretion (-31%), while markedly increasing plasma levels of total BAs (258%), cholic acid-derived BAs (295%), and chenodeoxycholic acid-derived BAs (217%), and decreasing the expression of hepatic genes involved in BA production. In subsequent experiments, mice were additionally treated with the BA sequestrant Colesevelam to inhibit BA reabsorption. Concomitant intestinal BA sequestration increased faecal BA excretion, normalized plasma BA levels, and reduced hepatic cholesterol. Moreover, concomitant BA sequestration further reduced plasma total cholesterol (-49%) and non-high-density lipoprotein cholesterol (-56%), tended to further attenuate atherosclerotic lesion area (-54%). Concomitant BA sequestration further increased the proportion of lesion-free valves (34%) and decreased the relative macrophage area within the lesion (-26%), thereby further increasing the plaque stability index (44%). Conclusion BA sequestration prevents the marked accumulation of plasma BAs as induced by prolonged brown fat activation, thereby further improving cholesterol metabolism and reducing atherosclerosis development. These data suggest that combining brown fat activation with BA sequestration is a promising new therapeutic strategy to reduce hyperlipidaemia and cardiovascular diseases

Novel role of a triglyceride-synthesizing enzyme:DGAT1 at the crossroad between triglyceride and cholesterol metabolism

AbstractAcyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1−/−) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1−/− and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia

Fecal Bile Salts and the Development of Necrotizing Enterocolitis in Preterm Infants

BACKGROUND: Intestinal bile salts (BSs) may be implicated in NEC development. We hypothesized that fecal BS levels are higher in preterm infants at risk for NEC. METHODS: We compared the composition and concentration of fecal BSs in ten preterm infants who developed NEC (Bell's Stage ≥ II) with twenty matched control infants without NEC. Conjugated and unconjugated fecal BSs were measured after birth (T1) and twice prior to NEC (T2, T3). Data are presented as medians and interquartile ranges. RESULTS: GA and BW were similar in all preterms: ~27+4 weeks and ~1010 g. Age of NEC onset was day 10 (8-24). T1 was collected 2 (1-3) days after birth. T2 and T3 were collected 5 (5-6) days and 1 (0-2) day before NEC or at corresponding postnatal ages in controls. The composition of conjugated BSs did not differ between the two groups. Total unconjugated BSs were 3-fold higher before NEC compared to controls at corresponding ages (0.41 μmol/g feces (0.21-0.74) versus 0.14 μmol/g feces (0.06-0.46), p < 0.05). CONCLUSION: Fecal BS concentrations are higher in preterm infants who develop NEC compared to infants without NEC. Further study is needed to determine the predictive value of fecal BSs in the development of NEC

Exercise Enhances Whole-Body Cholesterol Turnover in Mice

MEISSNER, M., R. HAVINGA, R. BOVERHOF, I. KEMA, A. K. GROEN, and F. KUIPERS. Exercise Enhances Whole-Body Cholesterol Turnover in Mice. Med. Sci. Sports Exerc., Vol. 42, No. 8, pp. 1460-1468, 2010. Purpose: Regular exercise reduces cardiovascular risk in humans by reducing cholesterol levels, but the underlying mechanisms have not been fully explored. Exercise might provoke changes in cholesterol and bile acid metabolism and thereby reduce cardiovascular risk. We examined whether voluntary wheel running in mice modulates cholesterol and bile acid metabolism. Methods: Male mice (10 wk old) were randomly assigned to have access to a voluntary running wheel for 2 wk (RUN group) or remained sedentary (SED group). Running wheel activity was recorded daily. In a first experiment, fecal sterol outputs, fecal bile acid profiles, plasma parameters, and expression levels of genes involved in cholesterol and bile acid metabolism were determined. In a second experiment, bile flow, biliary bile acid profile, and biliary secretion rates of cholesterol, phospholipids, and bile acids were determined. Results: The RUN group ran an average of 10 km.d(-1) and displayed lower plasma cholesterol compared with SED (P = 0.030). Fecal bile acid loss was induced by similar to 30% in running mice compared with SED (P = 0.0012). A similar to 30% increase in fecal cholesterol output in RUN (P = 0.014) was consistent with changes in parameters of cholesterol absorption, such as reduced plasma plant sterol-cholesterol ratio (P = 0.044) and decreased jejunal expression of Npc1l1 (P = 0.013). Supportive of an increased cholesterol synthesis to compensate for fecal sterol loss were increased hepatic mRNA levels of HMGCoA reductase (P = 0.006) and an increased plasma lathosterol-cholesterol ratio (P = 0.0011) in RUN. Conclusions: Voluntary wheel running increased cholesterol turnover in healthy mice owing to an increased fecal bile acid excretion and a decreased intestinal cholesterol absorption. Enhanced cholesterol turnover may contribute to the established reduction of cardiovascular risk induced by regular exercise

Developments in bile acid kinetic measurements using C-13 and H-2:10(5) times improved sensitivity during the last 40 years

Bile acid kinetics involve the measurement of pool sizes and turnover rates of individual bile acids. The technique is based on isotope dilution and was first described in the 1950s using radioactive C-14-labelled cholic acid (CA). It took until the 1970s before stable isotopes were introduced for this purpose (C-13, H-2) and isotope analysis methods were developed for CA and chenodeoxycholic acid (CDCA) applying gas chromatography/electron impact mass spectrometry. Until the 1980s, the isotope enrichment measurements were performed in bile samples aspirated from the duodenum. Thereafter, methodology became available allowing measurements to be performed in blood requiring at least 2 ml serum samples. Simultaneous measurement of kinetics of metabolically dependent CA and deoxycholic acid using C-13 and H-2 labels was introduced. Until the 1990s, this technique was only possible in adult humans due to the large sample sizes. Introduction of pentafluorobenzyl bromide derivatisation and electron capture negative ion mass spectrometry (GC/ECN-MS) reduced the sample volume to 50 mu l serum. This allowed isotope abundance measurement of CA in rats and in mice. However, repetitive collection of 100 mu l blood samples in mice is too invasive (collection via the orbita) and exhaustive. Therefore, the method development is now focussing on enhanced sensitivity and reduction of blank effects originating from the sample preparation. The final goal is to determine CA isotope enrichments in 20 mu l mouse blood obtained from the tail vein. This paper shows the feasibility of reaching this goal

Bile Acid Pool Dynamics in Progressive Familial lntrahepatic Cholestasis With Partial External Bile Diversion

Objectives: Partial external bile diversion (PEBD) is an established therapy for low-gamma-glutamyl transferase (GGT) progressive familial intrahepatic cholestasis (PFIC). This study sought to determine whether the dynamics of the cholic acid (CA) and chenodeoxycholic acid (CDCA) pools in subjects with low-GGT-PFIC with successful PEBD were equivalent to those achieved with successful liver transplantation (LTX). Methods: The kinetics of CA and CDCA metabolism were measured by stable isotope dilution in plasma samples in 5 subjects with PEBD, all with intact canalicular bile salt export pump expression and compared with subjects with low-GGT-PFIC with successful LTX. Stomal loss of bile acids was measured in subjects with PEBD. Results: The fractional turnover rate for CA in the PEBD group ranged from 0.5 to 4.2/day (LTX group, range 0.2-0.9/day, P = 0.076) and for CDCA from 0.7 to 4.5/day (LTX group 0.3-0.4/day, P = 0.009). The CA and CDCA pool sizes were equivalent between groups; however, pool composition in PEBD was somewhat more hydrophilic. The CA/CDCA ratio in PEBD ranged from 0.9 to 49.5, whereas in LTX it ranged from 0.5 to 2.6. Synthesis rates computed from isotope dilution correlated well with timed output for both CA (r(2) = 0.760, P = 0.024) and CDCA (r(2) = 0.690, P=0.021). Conclusions: PEBD results in bile acid fractional turnover rates greater than LTX, pool sizes equivalent to LTX, and pool composition that is at least as hydrophilic as produced by LTX

Dexamethasone exposure of neonatal rats modulates biliary lipid secretion and hepatic expression of genes controlling bile acid metabolism in adulthood without interfering with primary bile acid kinetics

Literature suggests that glucocorticoid (GC) exposure during early life may have long-term consequences into adult life. GCs are known to influence hepatic bile acid synthesis and their transport within the enterohepatic circulation. This study addresses effects of early postnatal exposure to GC on hepatic expression of key genes in bile acid metabolism and bile acid kinetics in adult rats. Male rats were treated with either dexamethasone (DEX) or saline at days 1-3 d after birth. Liver tissue and blood were collected from 2 d to 50 wk of age. Bile acid kinetics were determined at week 8. DEX acutely induced hepatic mRNA levels of cholesterol 7 alpha-hydroxylase (Cyp7a1), cholesterol 27-hydroxylase (Cyp27), and in particular sterol 12 alpha-hydroxylase (Cyp8b1), whereas expression of the bile acid transporters bile salt export pump (Bsep) and sodium taurocholate cotransporting polypeptide (Ntcp) was moderately affected. Neonatal DEX administration led to increased bilary lipid secretion, decreased Cyp8B1 mRNA expression and a 3-fold higher Cyp7a1/Cyp8b1 mRNA ratio in rats at week 8 compared with age-matched controls without alterations in bile acid kinetics. Therefore, neonatal DEX administration causes altered gene expressions later in life that are not translated into quantitative changes in bile acid kinetics