Simultaneous blood and biliary sampling of esculetin by microdialysis in the rat

Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To circumvent multiple blood sampling and interruption of enterohepatic circulation in conventional biliary cannulation, the present study utilized the minimally invasive sampling technique of microdialysis in pharmacokinetics and biliary excretion studies. Microdialysis probes were inserted into the jugular vein and bile duct in the anesthetized rat for simultaneous and continuous sampling following intravenous administration of esculetin, a bioactive coumarin derivative. Placements of the microdialysis probes were designed to minimize obstruction to normal flows of the body fluids. Separation and quantitation of esculetin in the dialysates were achieved using high performance liquid chromatography (HPLC) coupled to UV detection. The results indicated higher drug concentrations in the bile than in the blood, suggesting active biliary excretion. The study also provided an example of successful application of in vivo microdialysis as an interesting and feasible alternative for pharmacokinetics and biliary drug excretion studies.     

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.     

Effects of acute administration of taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.     

Increased Neopterin Levels in Brains of Patients with Human Immunodeficiency Virus Type 1 Infection

Postmortem levels of native neopterin (D-erythro-neopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1-infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus-infected patients, which frequently were more than threefold and occasionally more than 30-fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in approximately 25% of the human immunodeficiency virus type 1-infected patients was associated with enhanced levels of neopterin in frontal cortex.     

Biliary response to food in chickens with intact or interrupted enterohepatic circulation

The biliary secretion in response to food has been studied in chicken. In this species, a well defined biliary postprandial response with a clear vesicular component can be estimated. The bile salt independent fraction increases after feeding. Our results show that gallbladder bile has a lower osmotic power. The biliary response to food when the enterohepatic circulation is blocked, diminishes. The relationship between bile salt independent fraction values, whether the vesicular duct is tied or not, remains constant when the enterohepatic circulation is interrupted.     

Cerebrospinal Fluid Neopterin Analysis in Neuropediatric Patients: Establishment of a New Cut Off-Value for the Identification of Inflammatory-Immune Mediated Processes

Objective

A high level of cerebrospinal fluid (CSF) neopterin is a marker of central nervous system inflammatory-immune mediated processes. We aimed to assess data from 606 neuropediatric patients, describing the clinical and biochemical features of those neurological disorders presenting CSF neopterin values above a new cut-off value that was defined in our laboratory.

Methods

To establish the new CSF neopterin cut-off value, we studied two groups of patients: Group 1 comprised 68 patients with meningoencephalitis, and Group 2 comprised 52 children with a confirmed peripheral infection and no central nervous system involvement. We studied 606 CSF samples from neuropediatric patients who were classified into 3 groups: genetic diagnosis (A), acquired/unknown etiologic neurologic diseases (B) and inflammatory-immune mediated processes (C).

Results

The CSF neopterin cut-off value was 61 nmol/L. Out of 606 cases, 56 presented a CSF neopterin level above this value. Group C had significantly higher CSF neopterin, protein and leukocyte values than the other groups. Sixteen of twenty-three patients in this group had a CSF neopterin level above the cut-off, whereas three and seven patients presented increased leukocyte and protein values, respectively. A significant association was found among CSF neopterin, proteins and leukocytes in the 606 patients. White matter disturbances were associated with high CSF neopterin concentrations.

Conclusions

Although children with inflammatory-immune mediated processes presented higher CSF neopterin values, patients with other neurological disorders also showed increased CSF neopterin concentrations. These results stress the importance of CSF neopterin analysis for the identification of inflammatory-immune mediated processes.     

Influence of time, type of diet and ursodeoxycholic acid on biliary secretion in rats with intestinal resection

The effects of time and the type of dietary fat on biliary physiology in rats with 50% resection of the distal small intestine was investigated. The effects of ursodeoxycholic acid as an exogenous source in bile acid added to the diet were also studied. The fat composition of all diets was the same in quantitative terms (4%), and differed only in the type of lipid supplied: olive oil (diet A) or 1/3 medium chain triglycerides, 1/3 sunflower seed oil and 1/3 olive oil (diet B). Three months after intestinal resection, neither group of resected rats showed significant changes in bile flow or bile acid output in comparison to controls, whereas the slope of the regression line was notably increased, indicating that enterohepatic circulation failed to adapt to the different dietary fats during recovery from intestinal resection. The addition of ursodeoxycholic acid to diet B significantly raised bile acid flow and output in comparison to controls, as a result of the recovery of enterohepatic circulation. This was reflected in the lower slope of the regression line, thus suggesting that de novo synthesis in resected animals was less intensely stimulated.     

Identification of neopterin as a potential indicator of infection in burned patients.

Several fluorescent substances are present in the supernatants of acid-precipitated whole blood or plasma from burned patients. Perchloric acid supernatants of sera from infected, but not uninfected, burned rats contained a fluorescent substance with maximum emission at 420 nm at 355-nm excitation (355 ex/420 em). In this study of serum from burned human patients, several fluorescent substances were resolved by reverse-phase high-pressure liquid chromatography. One of these fluorescent components had an high-pressure liquid chromatography retention time and fluorescent characteristics identical to those of neopterin. The identification of this component as neopterin was verified by thermospray mass spectrometry. Serum neopterin concentrations were then determined in supernatants of patient serum samples having various levels of 355 ex/420 em fluorescence. A correlation was found between the concentrations of neopterin determined by high-pressure liquid chromatography and the presence of bacteremia in burned patients. These findings suggest that neopterin, which is a useful indicator of infection in other clinical settings, may also be an indicator of infection in burned patients.     

Pharmacokinetics, distribution and elimination of a synthetic octapeptide analogue of somatostatin in the rat

The in vivo fate of a long acting somatostatin analogue [des-AA1,2,3,4,13,14,D-Trp8,Gaba12]-somatostatin has been studied in the rat using biochemical and autoradiographic techniques. The analogue has a longer plasma half-life than somatostatin. This is due to its greater metabolic stability which renders it resistant to enzymic attack in the tissues. The primary route of elimination is by biliary excretion following clearance from the circulation by the liver. Evidence of enterohepatic circulation was found, but only to a very limited extent. When administered s.c., high plasma concentrations of intact peptide persist for a period of hours due to slow release from a stable depot at the injection site.     

Absorption and enterohepatic circulation of baicalin in rats

Pharmacokinetics of baicalin, in form of its parent drug (BG) and conjugated metabolites (BGM), were studied following intravenous and oral administration of baicalin to intact rats. The enterohepatic circulation of BG and BGM was also assessed in a linked-rat model. Multiple plasma and urine samples were collected, and concentrations of BG and BGM were determined using a liquid chromatography/tandem mass spectrometry method. The concentration of BGM was assayed in the form of baicalein after treatment with beta-glucuronidase/sulfatase. After i.v. administration, plasma concentration of BG rapidly declined with the elimination half-life (T1/2) of 0.1 till 4 h post dose, followed by slight increase from 4-8 h in plasma concentrations after drug administration. These plasma concentrations resulted in a significant prolongation of the terminal elimination half-life of BG (T1/2 TER, 9.7 h). BG also displayed slight increase in plasma concentrations (12-24 h) after oral administration, with T1/2 TER of 12.1 h. Based on the AUC of BG and BGM, the absolute bioavailability of baicalin was 2.2+/-0.2% and 27.8+/-5.6%, respectively. The exposure of baicalin to the systemic circulation was approximately 118-fold lower than that of BGM after oral administration (AUC0-t, 4.43 versus 523.97 nmol.h/mL). The high extent of glucuronidation suggested the possible presence of enterohepatic circulation, which was confirmed in the linked-rat model since plasma concentrations of BG and BGM were observed in bile-recipient rats at 4 to 36 h. The extent of enterohepatic circulation after intravenous administration of baicalin was 4.8% and 13.3% for BG and BGM, respectively. It was determined that 18.7% and 19.3% of the administered baicalin were subjected to enterohepatic circulation for BG and BGM, respectively, after oral administration. These results confirm that BG undergoes extensive first-pass glucuronidation and that enterohepatic circulation contributes significantly to the exposure of BG and BGM in rats.     

Asymptotics and bioavailability in a 17-compartment pharmacokinetic model with enterohepatic circulation and remetabolization

A 17-compartment linear pharmacokinetic model is designed, describing the complex process of enterohepatic circulation as a superposition of the net (remetabolizationfree) enterohepatic circulation, and remetabolization with subsequent intestinal absorption of the parent drug. Basically, the model is built by doubling the model describing the circulation of the parent drug in the body, so that the remetabolizable metabolite circulates in a model of the same structure as does the parent compound. The two submodels are cross-connected with arrows denoting the transition of the particular substance into the complementary part of the complex model. Asymptotic properties of the model are investigated, in particular, explicit formulas for its pharmacokinetic endpoints are given using the elements of its transition probability matrix. Conversely, taking account of the effect of bile cannulation, intravenous, intraportal and oral administration of the drug, as well as of the intravenous and intraportal administration of the remetabolizable metabolite, the transition probabilities of the system are determined in terms of certain measurable pharmacokinetic endpoints and the flow rates through the kidneys, liver and the cardiac output. Finally, the influence of the enterohepatic circulation and remetabolization process on bioavailability is examined. In particular, the inclusion-exclusion formula is derived, expressing its joint efficiency (defined as the relative increase of bioavailability) by means of the efficiencies of the net enterohepatic circulation and of the remetabolization process.     

Disposition and metabolism of 2-fluoro-beta-alanine conjugates of bile acids following secretion into bile

Since 2-fluoro-beta-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439-5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholyglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugated during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.     

Enterohepatic circulation of chloramphenicol and its glucuronide in the rat by microdialysis using a hepato-duodenal shunt

A system consisting of a hepato-duodenal shunt in which the bile of a drug-treated donor rat was diverted to the duodenum of an untreated recipient rat via a bile cannula was used to assess the role of hepatic metabolism and enterohepatic circulation in the pharmacokinetics of chloramphenicol. Blood concentrations of unbound chloramphenicol and its glucuronide were measured by on-line microdialysis coupled to a microbore liquid chromatographic system. Results indicated that chloramphenicol and its glucuronide were detected in the blood of both donor and recipient rats following an intravenous 100 mg/kg dose of chloramphenicol succinate to the donor rat. Our finding suggests that although enterohepatic circulation contributed only to a minor extent (approximately 1.8%) was involved in the disposition of unbound chloramphenicol in the rat on-line microdialysis techniques were applicable for such studies.     

Serum phenylalanine in patients post trauma and with sepsis correlate to neopterin concentrations

Increased blood concentrations of phenylalanine in patients with trauma and sepsis are common but unexplained. We examined the potential relationship between serum concentrations of phenylalanine and the immune activation marker neopterin in 84 specimens of 18 patients (14 males and 4 females) post-trauma during 12-14 days of follow up. Compared to healthy controls, average phenylalanine and neopterin concentrations were elevated in patients, and there existed a positive correlation between concentrations of the two analytes (r (s) = 0.375, p < 0.001). No such association existed between neopterin and tyrosine concentrations (r (s) = -0.018), but neopterin concentrations correlated to the phenylalanine to tyrosine ratio (r (s) = 0.328, p = 0.001). Increased phenylalanine implies insufficient conversion by phenylalanine (4)-hydroxylase (PAH). Oxidative stress due to immune activation and inflammation may destroy cofactor 5,6,7,8-tetrahydrobiopterin and impair PAH activity. This assumption is further supported by the correlation found between higher neopterin concentrations and higher phenylalanine to tyrosine ratio, which estimates efficacy of PAH.     

Biliary lipid secretion in hypercholesterolemia.

A report on the effects of primary bile acid ingestion alone or in combination with plant sterols on serum cholesterol levels, biliary lipid secretion, and bile acid metabolism. Biliary bile acid and cholesterol secretion were measured in four patients with type IIa hypercholesterolemia before and after randomized treatment periods. During these periods either a bile acid mixture (cholic-chenodeoxycholic 2:1, a proportion similar to that endogenously synthesized in health), at a level of 20 mg/kg, or the same mixture plus sitosterols, 200 mg/kg, was fed. Serum cholesterol and the cholesterol saturation of fasting-state bile was also measured. Pretreatment biliary lipid secretion was within normal limits. Bile acid kinetic measurements were also recorded before treatment and showed that cholic acid synthesis was disproportionately decreased relative to that of chenodeoxycholic acid, a finding previously reported by others. Administration of the bile acid mixture increased biliary bile acid secretion in 3 of 4 patients, but did not influence biliary cholesterol secretion. The combination of sitosterol-bile acid, however, caused a relative decrease in cholesterol secretion in bile, and fasting-state bile became unsaturated in all patients. No change in fecal neutral sterol excretion occurred during the beta-sitosterol-bile acid regimen, suggesting that simultaneous bile acid feeding blocks the compensatory increase in cholesterol synthesis known to be induced by beta-sitosterol feeding in hypercholesterolemic patients. Serum cholesterol levels also fell modestly during the sitosterol-bile acid regimen, the decrease averaging 15%. We conclude that the abnormally low rate of bile acid synthesis in patients with type IIa hyperlipoproteinemia does not influence biliary lipid secretion; that increasing the input of the two primary bile acids into the enterohepatic circulation does not increase biliary cholesterol secretion or lower serum cholesterol levels in such patients; and that the usual increase in cholesterol synthesis induced by beta-sitosterol feeding does not occur if bile acids are administered simultaneously.     

Metabolic fate of ethynodiol diacetate in the baboon.

The enterohepatic circulation and metabolism of ethynodiol diacetate (3beta,17beta-diacetoxy-17alpha-ethynyl-estr-4-ene) in baboons were studied following the intravenous injection of this contraceptive steroid labeled with 14C (4-position) and with 3H (in either the 3- or 17-acetoxy moieties). Bile and urine from four baboons with biliary fistulas and urine from four intact baboons were collected for 7 hours. On the average, 40% and 44% of the injected dose were excreted in the bile and urine, respectively. Only 48% was recovered in the urine of intact baboons. Analysis of these excretion rates indicates an insignificant enterohepatic circulation of this compound. The steroid was excreted mostly (over 80%) as a glucosiduronate in urine and bile. Very little excretion of the 3-acetoxy compound was detected in the urine or bile at any time interval. 17-Monoacetoxy compounds, however, were detected both in urine and bile, suggesting a difference in the rate of in vivo hydrolysis of the 17beta- vs. the 3beta-acetate.     

Bile-salt-dependent and independent choleresis induced by bucolome in the rat.

Choleresis induced by bucolome (BC) (1-cyclohexyl-5-n-butyl-2,4,6-trioxoperhydropyrimidine) was studied in male Wistar rats. [14C]Erythritol and mannitol clearance studies indicated this choleresis to be of canalicular origin. In 1-h continuous bile collection studies, immediately after the interruption of enterohepatic circulation (acute interruption), both bile flow and bile salt excretion rates were significantly increased in rats administered BC. However, the bile salt excretion rate fell rather rapidly in BC-administered rats, while the bile flow rate was fairly constant during this 1-h period. Thus, unlike the situation in control rats, bile flow rate was not significantly correlated with the bile salt excretion rate in BC-administered rats. In rats that had an external bile fistula open for 16-20 h (chronic interruption of enterohepatic circulation) the bile flow rate was also significantly increased by BC administration, while the bile salt excretion rate was not changed after BC administration. It is suggested that BC induced bile-salt-independent choleresis in both experimental rat groups (acute and chronic interruption of enterohepatic circulation). In addition, BC appeared to increase the bile-salt-dependent fraction of bile in rats with acute interruption of enterohepatic circulation, possibly by mobilizing the bile salt pooled in the intestinal content and (or) intestinal wall.     

Regulation of Bile Salt Pool Size in Man

When the enterohepatic circulation is intact the size of the bile salt pool is largely determined by the frequency of its enterohepatic circulation. This hypothesis, derived from studies of cholate kinetics in coeliac, cholecystectomy, and gall stone patients, represents the simplest interpretation of the data. A corollary is that daily cholate secretion is likely to be normal in these conditions and that therefore the propensity of bile to form cholesterol gall stones is not likely to be directly related to bile salt pool size.     

Disposition and metabolism of 2-fluoro-β-alanine conjugates of bile acids following secretion into bile

Since 2-fluoro-β-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439–5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholylglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugeted during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.     

Concentrations of neopterin and biopterin in the cerebrospinal fluid of patients with Parkinson's disease

The concentrations of neopterin and biopterin in CSF of 18 younger and 10 older, control patients and of 18 patients with Parkinson's disease were measured by high-performance liquid chromatography with fluorescence detection. Both neopterin concentrations and the neopterin to biopterin ratios in CSF were lower in 50-year or younger group than in 51-year or older group. Biopterin concentrations were also decreased but not significantly in the older group. The concentrations of neopterin and biopterin in CSF of patients with Parkinson's disease were lower than those of the age-matched older control group. However, the neopterin/biopterin ratios tended to be lower but not change significantly as compared to the age-matched older control group.