Selective inhibition of CYP27A1 and of chenodeoxycholic acid synthesis in cholestatic hamster liver

The aim of this study was to explore the regulation of serum cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in cholestatic hamster induced by ligation of the common bile duct for 48 h. The serum concentration of total bile acids and CA/CDCA ratio were significantly elevated, and the serum proportion of unconjugated bile acids to total bile acids was reduced in the cholestatic hamster similar to that in patients with obstructive jaundice. The hepatic CA/CDCA ratio increased from 3.6 to 11.0 (P<0.05) along with a 2.9-fold elevation in CA concentration (P<0.05) while the CDCA level remained unchanged. The hepatic mRNA and protein level as well as microsomal activity of the cholesterol 7alpha-hydroxylase, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase and 5beta-cholestane-3alpha,7alpha,12alpha-triol 25-hydroxylase were not significantly affected in cholestatic hamsters. In contrast, the mitochondrial activity and enzyme mass of the sterol 27-hydroxylase were significantly reduced, while its mRNA levels remained normal in bile duct-ligated hamster. In conclusion, bile acid biosynthetic pathway via mitochondrial sterol 27-hydroxylase was preferentially inhibited in bile duct-ligated hamsters. The suppression of CYP27A1 is, at least in part, responsible for the relative decreased production of CDCA and increased CA/CDCA ratio in the liver, bile and serum of cholestatic hamsters.     

Effect of dietary lipid and vitamin E on mitochondrial lipid peroxidation and hepatic injury in the bile duct-ligated rat.

To assess whether lipid peroxidation of hepatic mitochondria is associated with cholestatic hepatic injury we examined the effect of bile duct ligation (BDL) versus sham surgery on mitochondrial lipids of rats maintained on one of seven diets. Diets included vitamin E-deficient (E-) and vitamin E-sufficient (E+) combined with normal lipid (11.9% calories as stripped corn oil), high lipid (35% calories as stripped corn oil), or n-3 fatty acid (fish oil) supplementation. Rats were killed 17 days after surgery, mitochondria were isolated by differential centrifugation, and lipid-conjugated dienes and thiobarbituric acid-reacting substances (TBARS) were measured in mitochondrial lipids as indices of lipid peroxidation. BDL resulted in significant increases in lipid peroxidation in all dietary groups. The E- high lipid diets (with either corn oil or fish oil) were associated with higher lipid peroxide and serum bilirubin values in BDL rats compared to the normal lipid diets. Fish oil supplementation did not ameliorate cholestatic or oxidative injury. Serum alanine aminotransferase, bilirubin, alkaline phosphatase, and cholylglycine levels correlated significantly with levels of mitochondrial conjugated dienes and TBARS. These data suggest that free radical stress occurs during BDL in the rat and may result in mitochondrial lipid peroxidation, and that diets high in lipid may increase free radical damage to hepatic mitochondria. The role of free radicals in cholestatic hepatic injury requires further investigation.     

Effect of glucose ingestion on the metabolism of free fatty acids in human subjects

The rate of appearance of (14)CO(2) in expired air after the injection of a single dose of NaH(14)CO(3) has been determined in normal individuals both in the fasted and fed states. These data were combined with previously obtained results on the rate of disappearance of injected palmitate-(14)C from the bloodstream, to give a multicompartmental analysis of free fatty acid oxidation and esterification. The results confirm that glucose feeding promptly inhibits the rate of free fatty acid oxidation to CO(2). The "irreversible disposal rate," or irreversible flux of free fatty acids from the plasma, was also consistently reduced by glucose feeding. The diminution in irreversible disposal, not accounted for entirely by reduction of direct oxidation, must indicate suppression of other disposal mechanisms, including net esterification of free fatty acids. An average drop of 49% in "net esterification" when glucose was given may be compared with the 65% inhibition of rapid free fatty acid oxidation.     

Similarity of unusual bile acids in human umbilical cord blood and amniotic fluid from newborns and in sera and urine from adult patients with cholestatic liver diseases

Unusual bile acids in umbilical cord blood and amniotic fluid of term newborns and in sera and urine from adult patients with cholestatic liver diseases were analyzed by use of gas-liquid chromatography-mass spectrometry. These bile acids were compared in order to elucidate possible similarities of bile acid metabolism between fetal and cholestatic liver. In both umbilical cord blood and amniotic fluid, 14 unusual bile acids were found in addition to normal bile acids (cholic, chenodeoxycholic, deoxycholic, and lithocholic acids), and 15, excluding ursodeoxycholic acid, were found in sera and urine from patients with cholestatic liver diseases. Of the unusual bile acids detected, 12 were common to both samples. Six unusual bile acids, 3 beta-hydroxy- and 3 beta,12 alpha-dihydroxy-5-cholenoic acids, 3 alpha,6 alpha,7 alpha-trihydroxy-5 beta-cholanoic acid, 1 beta,3 alpha,12 alpha-trihydroxy-1 beta,3 alpha,7 alpha-trihydroxy-, and 1 beta,3 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholanoic acids were more abundant than others. They could be classified into three groups, i.e., unsaturated, 6-hydroxylated, and 1 beta-hydroxylated bile acids. 1 beta-Hydroxylated bile acids, which were not found in serum specimens, were detected in sera from umbilical cord blood and from patients with cholestatic liver diseases. The presence of these unusual bile acids suggested similarities between the altered metabolic states of the two groups examined.     

Bile acid production in human subjects: rate of oxidation of [24,25-3H]cholesterol compared to fecal bile acid excretion

Bile acid production has been quantitated in seven subjects by methods that compare the results of two independent approaches, namely, quantitation of cholesterol side-chain oxidation and fecal bile acid excretion. Six hypertriglyceridemic (HT) subjects and one normolipidemic control were studied by both techniques. A further control subject was studied by the cholesterol side-chain oxidation method alone. Cholesterol side-chain oxidation was quantitated by measuring the appearance of 3H2O after intravenous administration of [24,25-3H]cholesterol, using multicompartmental analysis of plasma cholesterol and [3H]water specific activity. Body water kinetics were independently defined by use of oral D2O. Two HT subjects were restudied while they were taking cholestyramine, 16 g/day. In all ten studies, multicompartmental analysis closely simulated the observed appearance of 3H2O. Values obtained for bile acid production suggest that cholesterol oxidation, or bile acid input, was significantly greater than fecal bile acid output in the HT subjects (P less than 0.05). Cholesterol side-chain oxidation rates in the two normal subjects were lower than those encountered in HT subjects, being similar to published values for normal subjects both for bile acid synthesis as determined by isotope dilution kinetics and fecal bile acid excretion. Studies conducted with two, synthetically different, preparations of [24,25-3H]cholesterol indicated that, in one of the two preparations, approximately 20% of the tritium label was at positions proximal to C24. In the other preparation examined, all of the tritium was located at, or distal to, C24. Further studies revealed that 0.055-0.24% of the dose was present as labile tritium by virtue of its appearance as 3H2O following in vitro incubation with human plasma. Provided these isotope effects are taken into account, multicompartmental analysis of plasma [24,25-3H]cholesterol and body water appears to be a useful technique for quantitating cholesterol oxidation in human subjects.     

Detection of 3-hydroxy-etianic and 3-hydroxy-bisnorcholanoic acids in human serum

Capillary GLC-MS analysis of the free/sulfate/glucuronide bile acid fraction obtained from human cholestatic serum demonstrated the presence of 3-hydroxyandrostan-17 beta-carboxylic (etianic) and 3-hydroxybisnorcholanoic acids.     

Characterization of different molecular forms of 5''-nucleotidase in normal serum and in serum from cholestatic patients and bile-duct-ligated rats.

Serum 5'-nucleotidase in rat and man is derived from the plasma membrane rather than the cytosol by the criteria of inhibition with [alpha beta-methylene]ADP and antisera. In individuals with cholestasis the serum enzyme is mainly present as a high-Mr form that in the presence of the zwitterionic detergent Sulphobetaine 14 has the electrophoretic characteristics of liver plasma-membrane ectoenzyme. A minor form of 5'-nucleotidase in cholestatic serum and all the enzyme in normal serum appears to be half the molecular size of the liver plasma-membrane ectoenzyme. 5'-Nucleotidase from both normal and cholestatic rat serum was found to contain a polypeptide chain of apparent Mr 70 000 by immunoblotting techniques. It is suggested that the major form of 5'-nucleotidase in cholestatic serum is an ectoenzyme dimer derived from liver plasma membrane. All of the enzyme in normal serum and some of the enzyme in cholestatic serum is present as an active monomer derived from the ectoenzyme dimer.     

Evaluation of glucose turnover, body mass and recycling with reversible and irreversible tracers

1. Methods are presented for the calculation of rates of synthesis or loss, mean transit time and total body pool of compounds from specific-radioactivity curves, without assuming a multicompartmental model and without fitting the data by exponential expressions. The methods apply to the steady state after either single injection or continuous infusion of a labelled compound. 2. The use of irreversible and reversible tracers and the effects of recycling of carbon on the estimations of the parameters of glucose metabolism are discussed. Methods for quantitatively determining recycling of glucose carbon by the use of glucose doubly labelled with (14)C and (3)H are presented.     

Staphylococcal bacteraemia,fusidic acid,and jaundice.

Fusidic acid was used to treat 131 out of 250 patients with staphylococcal bacteraemia over 10 years. Other antimicrobial agents were given to the 119 remaining patients. Thirty-seven patients were already jaundiced before antibiotic treatment was started. Jaundice developed during treatment in 38 out of 112 patients given fusidic acid (34%) and in two out of 101 patients given other antimicrobials. The incidence of jaundice was higher in patients given fusidic acid intravenously (48%) rather than by mouth (13%). Jaundice appeared within 48 hours after the administration of fusidic acid in 93% of these cases. When the drug was stopped serum bilirubin concentrations fell to normal values within four days in those patients in whom they had been previously normal and who survived the bacteraemic episode. Fusidic acid was associated with increasing jaundice in 13 of 19 patients (68%) already jaundiced before it was given. In six out of 32 patients who developed jaundice while receiving intravenous fusidic acid serum alkaline phosphatase activity was raised suggestive of cholestatic jaundice. The mechanism in the remaining patients was unknown. Fusidic acid, particularly the intravenous preparation, in invaluable in treating severe staphylococcal infection but should be used with caution in patients with abnormal liver function. Patients receiving intravenous fusidic acid should be given the oral form of the drug as soon as their clinical condition permits.     

无菌大鼠培育中无菌检测影响因素分析

目的对无菌大鼠进行无菌检测,分析其微生物污染的多种影响因素。方法通过采集无菌隔离器内环境、无菌大鼠及其粪便等标本,选用合适的培养基在不同的条件下对其进行培养,并于第7天和第14天转种于血平皿,以此来判断无菌动物的无菌状态,并分析出现微生物污染的影响因素。结果在被检的163例标本中,检出阳性13例,且各平行样检测结果一致。经复检确认,阳性结果中有10例是动物自身带菌,3例是培养基污染。结论本检测中所采用的培养基和方法准确可靠。检查用培养基质量、培养时间、标本处理等因素都可能影响无菌检测结果。     

In vivo metabolism of apolipoproteins A-IV and A-I associated with high density lipoprotein in normolipidemic subjects

The kinetics of apolipoprotein A-IV associated with high density lipoproteins (HDL) of plasma from fasting human subjects was followed for 15 days in five healthy normolipidemic volunteers. Purified apoA-IV and apoA-I were radioiodinated, respectively, with 125I and 131I, incubated in vitro with normal HDL, isolated at density 1.250 g/ml, and finally reinjected intravenously as HDL-125I-labeled apoA-IV and HDL-131I-labeled apoA-I. Blood samples were withdrawn at regular intervals for 15 days, and 24-h urine samples were collected. More than 93% (93.5 +/- 0.9%) of apoA-IV was recovered in apoA-I-containing lipoprotein particles after affinity chromatography on an anti-apoA-I column and 69.7 +/- 4.8% was bound to apoA-II in apoA-I:A-II particles separated on an anti-apoA-II column. 125I-labeled apoA-IV showed a much faster decay than 131I-labeled apoA-I for the first 5 days and thereafter the curves became parallel. Urinary/plasma ratios (U/P) for the 125I-labeled parallel. Urinary/plasma ratios (U/P) for the 125I-labeled apoA-IV were much higher than those for 131I-labeled apoA-I for the first days, but the U/P curves became parallel for the last 7 days, suggesting heterogeneity of apoA-IV metabolism. A heterogeneous multicompartmental model was constructed to describe the metabolism of lipoprotein particles containing apoA-IV and apoA-I and to calculate the kinetic parameters, fitting simultaneously all plasma and urine data for both tracers.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes

The relationships between cholesterol 7 alpha-hydroxylase activity, pool of free microsomal cholesterol, and degree of substrate saturation of the enzyme were studied in untreated (n = 5), cholesterol-fed (n = 4), and cholestyramine-treated (n = 6) gallstone patients undergoing cholecystectomy. Highly accurate methods based on isotope dilution-mass spectrometry were used for assay of the cholesterol 7 alpha-hydroxylase activity and for determination of the concentration of free cholesterol in the microsomes. The cholesterol-enriched diet increased the cholesterol 7 alpha-hydroxylase activity about twofold. Cholestyramine treatment was associated with a five- to sixfold increase of the cholesterol 7 alpha-hydroxylase activity. The concentration of free microsomal cholesterol remained essentially unchanged. The apparent degree of saturation of the enzyme was calculated to be 85% in the untreated patients, 86% in the cholesterol-fed patients, and 67% in those treated with cholestyramine. A significant negative correlation was obtained between enzyme activity and apparent substrate saturation. It is concluded that the apparent substrate saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes is high but that availability of cholesterol may limit the enzyme activity to some extent a high bile acid synthesis rates.     

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.     

Rapid synthesis and turnover of brain microsomal ether phospholipids in the adult rat.

The rates of synthesis, turnover, and half-lives were determined for brain microsomal ether phospholipids in the awake adult unanesthetized rat. A multicompartmental kinetic model of phospholipid metabolism, based on known pathways of synthesis, was applied to data generated by a 5 min intravenous infusion of [1,1-(3)H]hexadecanol. At 2 h post-infusion, 29%, 33%, and 31% of the total labeled brain phospholipid was found in the 1-O-alkyl-2-acyl-sn-glycero-3-phosphate, ethanolamine, and choline ether phospholipid fractions, respectively. Autoradiography and membrane fractionation showed that 3% of the net incorporated radiotracer was in myelin at 2 h, compared to 97% in gray matter microsomal and synaptosomal fractions. Based on evidence that ether phospholipid synthesis occurs in the microsomal membrane fraction, we calculated the synthesis rates of plasmanylcholine, plasmanylethanolamine, plasmenylethanolamine, and plasmenylcholine equal to 1.2, 9.3, 27.6, and 21.5 nmol. g(-1). min(-1), respectively. Therefore, 8% of the total brain ether phospholipids have half-lives of about 36.5, 26.7, 23.1, and 15.1 min, respectively. Furthermore, we clearly demonstrate that there are at least two pools of ether phospholipids in the adult rat brain. One is the static myelin pool with a slow rate of tracer incorporation and the other is a dynamic pool found in gray matter.The short half-lives of microsomal ether phospholipids and the rapid transfer to synaptosomes are consistent with evidence of the marked involvement of these lipids in brain signal transduction and synaptic function.     

A nonlinear kinetic model of γ-aminobutyric acid metabolism in a rabbit model of acute liver failure

To investigate the mechanisms by which serum levels of γ-aminobutyric acid (GABA) become elevated in experimental acute liver failure, a multicompartmental model of GABA metabolism has been constructed and used to simulate previously generated data on the kinetics of ³H-GABA uptake by isolated hepatocytes from normal rats and the kinetics of ³H-GABA in the plasma of normal rabbits, rabbits with galactosamine-induced acute liver failure, and rabbits with divascularized livers. Modeling analysis revealed that acute liver failure was associated with values for the mean fractional catabolic rate of GABA, plasma volume, and hepatic extraction of GABA that were 29%, 12%, and 49% less, respectively, than the corresponding control values. The defect in hepatic tissue extraction of GABA was sufficient to account for only 60% of the 10-fold increase in serum GABA levels that occurs in acute liver failure. Furthermore the 10-fold increase in serum GABA levels occured in acute liver failure before the onset of overt hepatic encephalopathy when hepatic extraction of GABA was not appreciably different from that found in normal rabbits. Thus the increase in serum GABA levels that occurs in acute liver failure cannot be attributed to a defect in hepatic extraction of GABA alone. Indeed, the modeling analysis indicated that in acute liver failure there is a 3—8-fold increase in the rate of delivery of GABA to the systemic circulation, but did not indicate its source.     

Continuous production of high degree casein hydrolysates by immobilized proteases in column reactor

The enzymatic complete hydrolysis of casein was investigated by using immobilized endopeptidase and exopepti dase packed in the jacketed column reactors. The mass transfer efficiency of proteins was improved by using sliced shrimp chitin hull as enzyme support, which formed a network structure inside the column reactor that prevented the formation of protein precipitate and increased the line flow rate of protein solution. The specificity of the protease was of crucial importance for both the hydrolysis degree and the free amino acid content of the hydrolysates. Of the enzymes tested, the immobilized A. oryzae protease was the most effective enzyme in breaking down the casein molecules and releasing the free amino acid from casein hydrolysates. The immobilized pancreatic and kidney exopeptidase could lead to a 20% increase of free amino acids. The free amino acid content of casein hydrolysates was 34.81% after processing and could reach to 64% if the column length was doubled, but 100% hydrolysis was impossible as the reverse reaction was also taking place. The casein hydrolysates was characterized by its high degree of hydrolysis and high content of free amino acids. It can be applied in infant formula, element diet, and as a protein ingredient for food industry.     

Biodegradation of phenanthrene in soil-slurry systems with different mass transfer regimes and soil contents

The effect of soil contents and mass transfer rates on soil bioremediation was investigated. Phenanthrene, a 3-ring polycyclic aromatic hydrocarbon (PAH), was chosen as a model target compound. The biodegradation tests were performed in soil-slurry systems at two distinct mass transfer rates: fast in flasks tests at 150 rpm and slow in roller-bottle tests at 2 rpm. The rate of phenanthrene biodegradation was similar at low soil content (2 wt.%) in both slurry systems, but the rates at high soil contents (6 and 18 wt.%) were higher in the roller-bottle tests. The maximum utilization rate constant for sorbed-phase biodegradation obtained from curve fitting using a mathematical model was decreased in the flask tests with increasing soil content, while not decreased in the roller-bottle tests.     

Quantitative aspects of free fatty acid metabolism in the fasted rat

Palmitate-1-(14)C was injected intravenously into unanesthetized, fasted rats. Disappearance of tracer from plasma free fatty acids was studied. A large component of free fatty acid (FFA) recycling was directly demonstrated by reinjection experiments. The latter studies also indicated the existence of an unidentified, rapidly turning over polar lipid in plasma which was synthesized from palmitate-(14)C. The appearance of (14)C in hepatic and extrahepatic triglycerides, in other esters, and in respired CO(2) was also followed. The data were analyzed using a multicompartmental model and a digital computer. Only a small fraction of the triglycerides formed in liver was derived directly from plasma free fatty acids. The major portion of net triglyceride formation appeared to be by way of an intermediate nontriglyceride ester pool which turned over relatively slowly compared to plasma free fatty acids. Initial approximations are as follows ( micromoles of fatty acid per min per 100 g body weight): net free fatty acid mobilization (irreversible disposal) = 2.4; hepatic triglyceride formation directly from plasma free fatty acid = 0.1; total hepatic lipid formation from plasma free fatty acids = 0.5; oxidation of free fatty acids to CO(2) = 0.8; percentage of respired CO(2) from direct oxidation of fatty acids = 12%; extrahepatic triglyceride formation directly from fatty acids = 0.4; total extrahepatic lipid formed directly from fatty acids = 1.2.     

Effect of parenteral nitrogen feeding on the free amino acid composition of the blood serum and liver tissue in irradiated rats

A considerable change in the free amino acid composition of blood serum and hepatic tissue was noted on the 7th and 14th days following total-body X-irradiation of rats with a dose of 2.9 Gy. The total free amino acid content of blood serum increased and that of hepatic tissue decreased by 85% (on an average) as compared to the intact controls. Quantitative changes in the content of individual amino acids were analysed. Polyamine injected enterally for 7 days and parenterally for 3 days after irradiation aids the elimination of the postirradiation changes in the amino acid balance.     

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.