High-cholesterol diet in combination with hydroxypropyl-beta-cyclodextrin induces NASH-like disorders in the liver of rats.

Non-alcoholic fatty liver disease (NAFLD) is a general term for fatty liver disease not caused by viruses or alcohol. Fibrotic hepatitis, cirrhosis, and hepatocellular carcinoma can develop. The recent increase in NAFLD incidence worldwide has stimulated drug development efforts. However, there is still no approved treatment. This may be due in part to the fact that non-alcoholic steatohepatitis (NASH) pathogenesis is very complex, and its mechanisms are not well understood. Studies with animals are very important for understanding the pathogenesis. Due to the close association between the establishment of human NASH pathology and metabolic syndrome, several animal models have been reported, especially in the context of overnutrition. In this study, we investigated the induction of NASH-like pathology by enhancing cholesterol absorption through treatment with hydroxypropyl-beta-cyclodextrin (CDX). Female Sprague-Dawley rats were fed a normal diet with normal water (control group); a high-fat (60 kcal%), cholesterol (1.25 %), and cholic acid (0.5 %) diet with normal water (HFCC group); or HFCC diet with 2 % CDX water (HFCC+CDX group) for 16 weeks. Compared to the control group, the HFCC and HFCC+CDX groups showed increased blood levels of total cholesterol, aspartate aminotransferase, and alanine aminotransferase. At autopsy, parameters related to hepatic lipid synthesis, oxidative stress, inflammation, and fibrosis were elevated, suggesting the development of NAFLD/NASH. Elevated levels of endoplasmic reticulum stress-related genes were evident in the HFCC+CDX group. In the novel rat model, excessive cholesterol intake and accelerated absorption contributed to NAFLD/NASH pathogenesis.

[Successful implantation of a metal coronary angioplasty stent for bronchomalacia of the right tracheal bronchus associated with right isomerism complex in an early infant].

One-month-old boy had severe emphysema of the right upper lobe due to the stenotic tracheal bronchus compressed between the distorted right patent ductus arteriosus (PDA) and the right aortic arch associated with right isomerism complex. He underwent a left modified Blalock-Taussig shunt and a division of the PDA on cardiopulmonary bypass. Extracorporeal lung support (ECLS) was introduced because of severe hypoxemia caused by remaining bronchomalacia of the tracheal bronchus. On postoperative day 3, a metal coronary angioplasty stent was implanted at the stenotic lesion under fluoroscopic and bronchoscopic guidance. He was successfully weaned from ECLS and then respirator after the implantation. This simple stenting procedure might be an effective alternate in the treatment of bronchomalacia or bronchial stenosis in early infancy.

Thiolase involved in bile acid formation.

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.

A direct enzyme immunoassay for 18-hydroxycortisol in urine: a new tool for screening primary aldosteronism.

A microplate enzyme immunoassay has been developed for the measurement of 18-hydroxycortisol in urine. An antiserum was produced by immunization of rabbits with a 3-O-(carboxymethyl)oximino-18-hydroxycortisol-bovine serum albumin conjugate. IgG was isolated from the antiserum and was biotinylated. Newly synthesized p-nitrophenyl ester of the oxime was used for the preparation of steroid-horseradish peroxidase conjugate. After an incubation of diluted urine samples (or standards) and the steroid-enzyme conjugate with the biotinylated antibody, the resulting antigen-antibody complex was separated by adding a portion of the reaction mixture into the avidin-coated microtiter plate. Peroxidase bound to solid phase was measured colorimetrically. The standard curve was linear from 0.25 to 10 ng/well. Intra- and interassay coefficients of variation were 5.5-8.8 and 7.8-8.2%, respectively. The assay was specific except for 18-hydroxycortisone with minor cross reaction. Urinary 18-hydroxycortisol excretion ranged 836-7460 and 26-696 nmol/24 h, respectively, in patients with primary aldosteronism (n = 8) and in control subjects (n = 40). This simple and rapid (< 4 h) assay is suitable for screening patients with primary aldosteronism.

Monoclonal antibodies specific for 18-hydroxycortisol and their use in an enzyme immunoassay for human urinary 18-hydroxycortisol for diagnosis of primary aldosteronism.

We report the development of a monoclonal antibody-based enzyme immunoassay (EIA) specific for human urinary 18-hydroxycortisol, a biological marker of primary aldosteronism. Hybrid cell lines (hybridomas) were isolated after fusion between myeloma cells and spleen cells prepared from mice immunized with 18-hydroxycortisol conjugate. A competitive EIA suitable for the measurement of urinary 18-hydroxycortisol was developed using the mouse monoclonal antibody, KTM-41, which showed no practical cross-reaction with related endogenous steroids and synthetic steroids. This EIA meets all the requirements of routine clinical assay in terms of sensitivity (detection limit: 20 nmol/L), reproducibility (total CV: 8-15%), accuracy (recovery: 88-115%), simplicity and rapidity (< 3 h). Urinary 18-hydroxycortisol measured by the present assay was 153 +/- 119 nmol/L (mean +/- SD, range, 28-485) and 1787 +/- 1180 (range, 810-4264) in normal subjects (n = 20) and in patients with primary aldosteronism (n = 7), respectively. Clinical validation of the assay was confirmed by an appropriate decrease in urinary 18-hydroxycortisol level in patients with primary aldosteronism subsequent to adrenalectomy: 171 +/- 141 nmol/L (range, 41-466).

Synthesis of 4- and 16 alpha-hydroxylated equine estrogens.

4-Hydroxyequilin, 4-hydroxyequilenin, and 16 alpha-hydroxyequilenin were synthesized as authentic specimens for the metabolic studies of equine estrogens. The synthetic route leading to the 4-hydroxylated compounds was started from o-vanillin, which was transformed into the beta-ketosulfoxide (2b) by sequential multistep reactions. This was converted to the alpha,beta-unsaturated ketone (3) as Michael acceptor. Condensation of 3 with 2-methylcyclopentane-1,3-dione, followed by ring closure with methanesulfonic acid provided the cyclized estrapentaene (5). Several oxidoreduction reactions were then performed to give the desired compounds. Preparation of 16 alpha-hydroxyequilenin was attained by reductive cleavage of the 16 alpha,17 alpha-epoxide formed from equilenin.

Synthesis of diastereomers of 3 alpha,7 alpha,12 alpha, 24-tetrahydroxy- and 3 alpha,7 alpha,24-trihydroxy-5 beta-cholestan- 26-oic acids and their structures.

Four stereoisomers of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acids were synthesized as possible intermediates of the side-chain degradation step of bile acid biosynthesis. 3 alpha,7 alpha,12 alpha-Trihydroxy-5 beta-cholest-25-en-24-one prepared by thermolysis of beta-ketosulfoxide was reduced to the (24R)- and (24S)-allylic alcohols by reduction with sodium borohydride. Each isomeric alcohol was subjected to hydroboration and oxidation to give (25R)- and (25S)-3 alpha,7 alpha,12 alpha,24,26-pentahydroxy-5 beta-cholestanes. The separated four stereoisomers were converted into the corresponding 26-carboxylic acids. The stereoisomers of 3 alpha,7 alpha,24-trihydroxy-5 beta-cholestan-26-oic acids were synthesized in the same manner. To establish the stereochemistry of these carboxylic acids, the chemical transformation of methyl 3 alpha,7 alpha,12 alpha-trihydroxy- and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-en-26-oates into the above stereoisomers and the reductive dehydroxylation of the 24-hydroxyl group into known 3 alpha,7 alpha,12 alpha,26-tetrahydroxy- and 3 alpha,7 alpha,26-trihydroxy-5 beta-cholestanes are described. The applications of spectroscopic methods (circular dichroism and 1H nuclear magnetic resonance) to elucidation of the stereochemistry are also discussed.

Synthesis of 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids by the use of beta-ketosulfoxide.

The biosynthetic intermediates of bile acid, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acids, were synthesized by means of the thermal elimination of beta-ketosulfoxides. The alpha, beta-unsaturated ketones as key compounds of the synthesis, 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha-dihydroxy-5 beta-cholest-25-en-24-ones, were effectively derived from the beta-ketosulfoxides prepared from methyl cholate or chenodeoxycholate by reaction with methylsulfinylcarbanion. These unsaturated ketones were converted into 3 alpha, 7 alpha, 12 alpha, 26-tetrahydroxy- and 3 alpha, 7 alpha, 26-trihydroxy-5 beta-cholestanes by reductive deoxygenation and hydroboration, of which stereoisomers were chromatographically separated into 25S- and 25R- isomers. The oxidation of each of the above isomeric alcohols after the protection of the hydroxyl groups on the steroidal ring and the following hydrolysis gave the title 26-carboxylic acids.

A convenient synthesis of 3 beta,12 alpha-, 3 beta,7 alpha-, and 3 beta,7 beta-dihydroxy-5-cholen-24-oic acids: unusual bile acids in human biological fluids.

The unusual bile acids 3 beta,12 alpha- (V), 3 beta,7 alpha- (XIIIa), and 3 beta,7 beta- (XIIIb) dihydroxy-5-cholen-24-oic acids were synthesized conveniently from the 3-oxo derivatives of deoxycholic (I) and lithocholic (VI) acids, respectively, to provide authentic samples for the gas chromatography-mass spectrometric determination of these bile acids in the abnormal metabolism of bile acids.

An efficient synthesis of 4 beta- and 6 alpha-hydroxylated bile acids.

An efficient method for the preparation of 4 beta- and 6 alpha-hydroxylated bile acids has been developed. It involved a highly stereoselective acetoxylation at the 4 beta and 6 alpha positions of 3- and 7-oxo bile acids, respectively, with lead tetraacetate in the presence of boron trifluoride etherate in acetic acid. Reduction of the resulting alpha-acetoxy ketones with sodium borohydride or tert-butylamine borane complex, and alkaline hydrolysis, provided the desired bile acids in good yields.

Determination of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate in human serum by gas chromatography-mass spectrometry.

The glycine conjugate of 3 beta-hydroxy-5-cholen-24-oic acid and its sulfate labeled with deuterium at the C-2, -4, and -23 positions were synthesized. A highly sensitive and specific quantitative assay of the bile acid has been developed by selected ion monitoring in gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives using the deuterium labeled conjugates as internal standards. Calibration curves for the bile acid and its sulfate exhibited a linear relationship over the range of 0.01-100 micrograms/ml in human serum.

Convenient synthesis of 18-hydroxylated cortisol and prednisolone.

18,20-Epoxy-11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one was synthesized by the application of hypoiodite reaction to the cortisol acetonide. The intermediary 18-iodo derivative was converted to the 11-oxo steroid by chromic acid prior to silver ion-assisted solvolysis. Removal of the protective group with hydrochloric acid was finally carried out to give the desired 11 beta,17 alpha,18,21-tetrahydroxypregn-4-ene-3,20-dione as the hemiacetal form. 18,20-Epoxy-11 beta-17 alpha,20 beta,21- tetrahydroxypregna-1,4-dien-3-one was also prepared from prednisolone through a similar reaction sequence.

Effect of the hydroxyl group on the oxidative cleavage (beta-oxidation) of steroidal side chain for bile acid biosynthesis in rat liver homogenate.

Mono-, di-, tri-, and tetrahydroxy-5 beta-cholestan-26-oic acids were incubated with rat liver homogenate (800 x g supernatant and light mitochondrial fraction) to study substrate specificity in the side-chain cleavage reaction (beta-oxidation) of bile acid biosynthesis. The C27-intermediates (5 beta-cholest-24-en-26-oic acids and 24-hydroxy-5 beta-cholestan-26-oic acids) in beta-oxidation and the corresponding C24-bile acids were quantitatively determined by capillary gas chromatography. Monohydroxy-5 beta-cholestan-26-oic acid was not converted into C24-bile acid. Di- and trihydroxy-5 beta-cholestan-26-oic acids were effectively transformed into the C27-intermediates and C24-bile acids. Tetrahydroxy-5 beta-cholestan-26-oic acids were also converted into C27-intermediates and corresponding C24-bile acids. The intermediate 24-hydroxy-5 beta-cholestan-26-oic acids could not be detected in the products by incubation with the light mitochondrial fraction. The total specific activity of protein in the light mitochondrial fraction for the production of C27-intermediates and C24-bile acids was higher than that of 800 x g supernatant solution. The effects of the number and the position of hydroxyl groups on the side-chain degradation are discussed.

The LEC (Long-Evans Cinnamon) rat as an animal model for bilirubin-induced tooth pigmentation.

The LEC (Long-Evans Cinnamon) rat is well known as a useful animal model for hepatic disease. We noticed the green pigmentation in incisors 2-3 weeks after acute hepatitis accompanied by severe jaundice. This study was undertaken to elucidate the cause of this phenomenon. Half of the pigmented teeth were examined by histopathological analysis and microradiographic analysis. Pigmentation was observed as a green stripe that ran parallel to the incremental line in the dentine. The microradiographic analysis disclosed enhanced permeability of the pigmented area as compared with other areas. The rest of pigmented teeth were dried, powdered and bilirubin was extracted with chloroform /methanol/acetic acid, 30:10:0.5; v/v under sonication. After centrifugation, the supernatant was collected and evaporated. The residue was dissolved in chloroform and its absorption spectrum measured after diazo reaction to reveal the presence of bilirubin. The spectral characteristics indicated the presence of bilirubin in the pigmented teeth. Thus, the LEC rat may be useful animal model for bilirubin-induced tooth pigmentation.

Developmental pattern of 3-oxo-delta 4 bile acids in neonatal bile acid metabolism.

AIMS: To investigate whether a fetal pathway of bile acid synthesis persists in neonates and infants. METHODS: 3-oxo-delta 4 bile acids were determined qualitatively and quantitatively in the urine, meconium, and faeces of healthy neonates and infants, using gas chromatography-mass spectrometry. RESULTS: The mean percentage of 3-oxo-delta 4 bile acids in total bile acids in urine at birth was significantly higher than that at 3 or 7 days, and at 1 or 3 months of age. The concentration of this component in meconium was significantly higher than that in faeces at 7 days and at 1 or 3 months of age. CONCLUSIONS: The presence of large amounts of urinary 3-oxo-delta 4 bile acids may indicate immaturity in the activity of hepatic 3-oxo-delta 4-steroid 5 beta-reductase in the first week of postnatal life. Large amounts of this component in meconium may be due to the ingestion of amniotic fluid by the fetus during pregnancy.

Experimental bilirubin pigmentation of rat dentine and its detection by a qualitative analytical method.

An animal model of bilirubinemia was used to determine whether bilirubin present in pigmented teeth can be extracted and qualitatively analysed. The bile ducts of 10 Long-Evans Agouti rats were ligated and bilirubin (14 mg/kg per day) was injected intraperitoneally for 4 days. When the animals were killed 2 weeks later, pigmented lower incisors were observed in three animals. These teeth were dried, powdered and bilirubin was extracted with chloroform/methanol/acetic acid, 30:10:0.5, v/v for 10 min under sonication. After centrifugation, the supernatant was collected and evaporated. The residue was dissolved in chloroform and its absorption spectrum measured before and after diazo reaction. This resulted in a shift of the absorption maximum from 450 to 540 nm and indicated the presence of bilirubin in pigmented teeth. No bilirubin was found in the lower incisors of untreated control rats. This technique may be useful in distinguishing bilirubin staning from other intrinsic discolorations of teeth.

Analysis of stereoisomeric C27-bile acids by high performance liquid chromatography with fluorescence detection.

A method for differentially measuring the 24-hydroxylated stereoisomeric intermediates (3 alpha,7 alpha,12 alpha,24-tetrahydroxy- and 3 alpha,7 alpha,24-trihydroxy-5 beta-cholestan-26-oic acids) and related C27-bile acids in beta-oxidation of bile acid biosynthesis has been developed by high performance liquid chromatography with fluorescence detection. The method involved the derivatization of the above intermediable C27-bile acids into fluorescent esters with 3-(4-bromomethylphenyl)-7-diethylaminocoumarin, a newly synthesized labeling reagent for carboxylic acids. The fluorescent derivatives were subjected to a short silica gel column to eliminate interfering products prior to analysis by high performance liquid chromatography. The separation of the 16 kinds of bile acids containing stereoisomers was carried out using a reversed-phase Inertsil C8-column by gradient elution and detected with a fluorometer (Ex. 400 nm, Em. 475 nm). The linearity of calibration curve for each bile acid was from 0.5 to 250 pmol (r = 0.999) and the detection limits were about 15 fmol at a signal-to-noise ratio of 3. The method was applied to the determination of intermediates in beta-oxidation of bile acid biosynthesis using rat liver homogenate. The results showed that two stereoisomers of 24-hydroxylated C27-bile acids were predominantly produced, indicating the formation of the isomers by the cis-hydration with water.

Large amounts of 1 beta-hydroxylated bile acids in urine during taurine therapy.

The management of infants with cholestasis remains a difficult challenge. On the hypothesis that taurine is effective in treating neonatal cholestasis, taurine (1 g/day, per os) was administered to 2 patients with neonatal hepatitis and the bile acids were analyzed using gas chromatography-mass spectrometry (GC-MS). The serum levels of bilirubin and bile acids were significantly decreased by taurine. Before the treatment, cholic acid (CA) and chenodeoxycholic acid (CDCA) were predominant (79.2% in both patients) in the urine. There was a significant elevation of 1 beta-hydroxylated bile acids (1 beta BA), especially 1 beta, 3 alpha, 7 alpha, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid (CA-1 beta-ol), in urine collected during the taurine therapy, and 1 beta BA became predominant (57.7-78.3%). Therefore, increased amounts of urine 1 beta BA were excreted during taurine administration. Taurine therapy is recommended, because it might be effective for treating neonatal cholestasis.

[Determination of fetal bile acids and related steroidal compounds and their profile in neonatal biological fluids].

The unusual bile acids hydroxylated at 1 beta-, 2 beta-, 4 beta-, 6 alpha- and 19-positions of cholic and chenodeoxycholic acids have been found from the meconium, neonatal bile, blood and urine, and amniotic fluid and pregnant urine by GC-MS analysis. These hydroxylated bile acids and their conjugates were synthesized as their references from the corresponding usual bile acids as starting materials, and the simultaneous and high performance analytical methods were developed by GC-MS, HPLC and enzyme immunoassay. The above mentioned unusual bile acids were identified and determined in significant amounts of the total bile acids in the biological fluids from neonates and pregnant women, but not from normal adults. We, therefore, proposed that they should be called as "fetal bile acids". Application of the developed methods was performed for the studies on the dynamic profile of fetal bile acids in developing fetus and neonates, and the clinical diagnosis of the hepatobiliary diseases of infants and congenital bile acid biosynthetic disorders, Zellweger syndrome, celebrotendinus xanthomatosis, 3-oxo-delta 4-steroid 5 beta-reductase deficiency and congenital biliary atresia. Analyses of steroidal hormones, equine estrogens and 18-hydroxycortisol were also described.

[Radioimmunological characterization of anti lithocholic acid antisera elicited by [C-6] carboxylic acid N-succinimidyl esters as haptenic derivatives].

In order to investigate the antigenic effects of the carboxyl group in the side chain and the bridge length on producing anti lithocholic acid antibody, the immunogens in which bile acid is coupled with bovine serum albumin through propionyloxy or butyryloxy amide linkage at the C-6 position were prepared. The antibodies elicited by these conjugates had low titers but showed a high affinity for lithocholic acid with association constants in the range of 0.42-1.04 X 10(8)M-1. The cross-reactivities of both antisera were essentially the same as that of methyl 6 alpha-hemisuccinyloxylithocholate. These results suggested that the side chain hydrolysis product of the methylated antigen worked as a real antigen in the body.