ABSTRACT
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.
Subject(s)
Hypercholesterolemia , Hyperlipidemias , Non-alcoholic Fatty Liver Disease , Humans , Rats , Female , Animals , Non-alcoholic Fatty Liver Disease/chemically induced , 2-Hydroxypropyl-beta-cyclodextrin/metabolism , 2-Hydroxypropyl-beta-cyclodextrin/therapeutic use , Rats, Sprague-Dawley , Diet, High-Fat/adverse effects , Liver/metabolism , Cholesterol , Hypercholesterolemia/metabolism , Disease Models, AnimalABSTRACT
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.
Subject(s)
Angioplasty , Bronchial Diseases/surgery , Coronary Vessels/surgery , Heart Defects, Congenital , Stents , Tracheal Stenosis/surgery , Bronchial Diseases/pathology , Cardiac Surgical Procedures/methods , Constriction, Pathologic , Ductus Arteriosus, Patent/complications , Heart Defects, Congenital/complications , Humans , Infant , Male , Spleen/abnormalitiesABSTRACT
A microplate enzyme immunoassay (EIA) was developed for the measurement of glycine- and taurine-conjugated 7alpha-hydroxy-3-oxo-4-cholenoic acids (CDCA-delta4-3-one) in human urine. The antiserum was prepared by immunizing rabbits with N-(7alpha-hydroxy-3-oxo-4-cholen-24-oyl)-3-aminopropionic acid--bovine serum albumin conjugate. A colorimetric EIA was established using horseradish peroxidase-labeled antigen having a shorter bridge length than that of the immunogen, and 3, 3', 5, 5'-tetramethylbenzidine /hydrogen peroxide for the measurement of the enzyme activity. The reactivities of the antiserum for glycine and taurine conjugates of CDCA-delta4-3-one was almost the same. The specificity of the antiserum was investigated by determining the cross-reactivities of various bile acids and related compounds. An appropriate dose-response curve for conjugated CDCA-delta4-3-one was obtained in the range of 0.05-10 ng/well. This method was used for direct analysis of conjugated CDCA-delta4-3-one in urine of healthy infants and patients with liver diseases.
Subject(s)
Chenodeoxycholic Acid/analogs & derivatives , Chenodeoxycholic Acid/urine , Chenodeoxycholic Acid/chemistry , Cross Reactions , Haptens/chemistry , Humans , Immune Sera , Immunoenzyme Techniques , Nuclear Magnetic Resonance, Biomolecular , Sensitivity and Specificity , Spectrophotometry, InfraredABSTRACT
Unusual bile acids, such as unsaturated ketonic and 7beta-hydroxylated bile acids, have been detected in urine early in life. To elucidate the normal profiles of usual and unusual urinary bile acids in the neonatal and pediatric periods, we measured the concentrations of 28 kinds in urine from normal newborns, infants, and children by gas chromatography-mass spectrometry. The mean total bile acid/Cr ratio in 7-d-old infants was significantly higher than in subjects of other age groups (birth, 2-4 mo, 5-7 mo, 11-12 mo, 2-3 y, 9-14 y, and adult) (p < 0.05). Relatively large amounts of unusual bile acids were detected during infancy, especially during the period up to 1 mo of age. At that time, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5bet a-cholan-24-oic, 7alpha, 12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic, and 7alpha,12alpha-dihydroxy-3-oxo-4-cholen-24-oic acids were predominant among the unusual urinary bile acids present. Moreover, the levels of 3alpha,7beta,12alpha-trihydroxy-5beta-cholan+ ++-24-oic acid increased significantly after 2-4 mo of age. These results indicate that bile acid synthesis and metabolism in the liver of developing infants are significantly different from that occurring in the liver of adults. Significant amounts of urinary isomerized 7beta-hydroxylated bile acids were detected after late infancy, probably because of changes in the intestinal bacterial flora response to a change in nutrition. We describe, for the first time, evidence of the epimerization of the 7alpha-hydroxyl group of cholic acid, which may be unique to human development.
Subject(s)
Aging/urine , Bile Acids and Salts/metabolism , Bile Acids and Salts/urine , Adolescent , Adult , Bile Acids and Salts/biosynthesis , Child , Child, Preschool , Humans , Hydroxylation , Infant , Infant, Newborn , Ketones , Liver/growth & development , Liver/metabolismSubject(s)
Bile Acids and Salts/urine , Cholestasis/urine , Jaundice, Neonatal/urine , Alanine Transaminase/blood , Bilirubin/blood , Child, Preschool , Cholestasis/blood , Cholestasis/etiology , Female , Gas Chromatography-Mass Spectrometry , Humans , Infant , Infant, Newborn , Jaundice, Neonatal/blood , Jaundice, Neonatal/etiology , Statistics, Nonparametric , Tyrosine/blood , gamma-Glutamyltransferase/bloodABSTRACT
BACKGROUND: It has been suggested that quantitative analysis of urinary bile acids may help to distinguish primary 3-oxo-delta 4-steroid 5 beta-reductase deficiency from the excretion of 3-oxo-delta 4 bile acids that occurs as a result of liver damage. METHODS: Urinary bile acids were quantitatively analyzed by gas chromatography-mass spectrometry in four Japanese patients with severe neonatal cholestasis associated with hypertyrosinemia without urinary succinylacetone (i.e. tyrosinemia type I-like disease). These four patients represented sporadic cases. RESULTS: Large amounts of 3-oxo-delta 4 bile acids were detected, which comprised greater than 80% of the total urinary bile acids. Small amounts of allo-bile acids and primary bile acids were also detected, comprising less than 1% and 15% of the total urinary bile acids, respectively. CONCLUSIONS: It was suspected that these four patients had a primary 3-oxo-delta 4-steroid 5 beta-reductase deficiency. However, it is possible that some patients in this study may have had a secondary 3-oxo-delta 4-steroid 5 beta-reductase deficiency, caused by idiopathic neonatal cholestatic liver failure.
Subject(s)
Amino Acid Metabolism, Inborn Errors/diagnosis , Bile Acids and Salts/urine , Cholestasis/urine , Oxidoreductases/deficiency , Tyrosine/blood , Amino Acid Metabolism, Inborn Errors/urine , Cholestasis/complications , Diagnosis, Differential , Female , Humans , Infant , Male , Tyrosine/urineABSTRACT
A stroma-dependent cell line (HB-1) was established from myelogenous leukemic cells of CBA/N mouse. Characterization of the cells showed that HB-1 proliferated on hematopoietic supportive stromal cells (MS-10), but did not survive or proliferate on hematopoietic nonsupportive cells (MS-K). Direct contact between HB-1 and MS-10 appears to be necessary for HB-1 to proliferate on MS-10. We found that interleukin-1alpha (IL-1alpha) produced by MS-10 plays a major role in the survival and proliferation of HB-1. IL-11 did not support the proliferation of HB-1 cells by itself, but enhanced the proliferation of HB-1 cells in the presence of IL-1alpha. The expression of IL-1alpha and IL-11 was induced in MS-10 by the direct contact with HB-1 cells, and the expression of IL-1 receptor type I (IL-1RI) and interleukin-11 receptor (IL-11R) was induced in HB-1 cells by the attachment of the cells to MS-10. These findings show the existence of two-way interactions between HB-1 and MS-10.
Subject(s)
Bone Marrow Cells/cytology , Cell Communication , Hematopoietic Cell Growth Factors/pharmacology , Hematopoietic Stem Cells/cytology , Interleukin-11/physiology , Interleukin-1/physiology , Neoplastic Stem Cells/cytology , Receptors, Interleukin-1/physiology , Receptors, Interleukin/physiology , Acute Disease , Animals , Antibodies, Monoclonal/pharmacology , Bone Marrow Cells/metabolism , Cell Differentiation/drug effects , Cell Division/drug effects , Culture Media, Conditioned/pharmacology , Female , Gene Expression Regulation/drug effects , Hematopoietic Cell Growth Factors/biosynthesis , Hematopoietic Cell Growth Factors/genetics , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Interleukin-1/biosynthesis , Interleukin-1/genetics , Interleukin-1/pharmacology , Interleukin-11/biosynthesis , Interleukin-11/genetics , Interleukin-11/pharmacology , Interleukin-11 Receptor alpha Subunit , Leukemia, Myeloid/pathology , Leukemia, Radiation-Induced/pathology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Polymerase Chain Reaction , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/genetics , Receptors, Interleukin-1/biosynthesis , Receptors, Interleukin-1/genetics , Receptors, Interleukin-11 , Recombinant Proteins/pharmacology , Stromal Cells/metabolismABSTRACT
UNLABELLED: A 3-oxo-delta4-steroid 5beta-reductase (5beta-reductase) deficiency is difficult to diagnose because severe liver damage can result in a similar pattern of metabolite excretion. We investigated the usefulness of immunoblot analysis for diagnosis of 5beta-reductase deficiency and quantitatively analysed urinary bile acids by gas chromatography-mass spectrometry in a 5-month-old Japanese boy with severe neonatal cholestasis associated with hypertyrosinaemia. A liver sample was examined by immunoblot analysis using monoclonal antibodies against 5beta-reductase. Urinary 3-oxo-delta4 bile acids accounted for 88.3% of total bile acids, 5alpha-bile acids for 0.9%, and primary bile acids for 9.1%. Immunoblot analysis of the liver tissue showed an indistinct band of 5beta-reductase. CONCLUSIONS: These findings suggest that this patient had a secondary 5beta-reductase deficiency due to severe liver damage, even though 3-oxo-delta4 bile acids constituted more than 70% of total urinary bile acids. However, the patient may possibly have had an inherited 5beta-reductase deficiency.
Subject(s)
Liver/pathology , Metabolism, Inborn Errors/diagnosis , Oxidoreductases/deficiency , Amino Acid Metabolism, Inborn Errors/complications , Bile Acids and Salts/urine , Biopsy , Cholestasis/complications , Gas Chromatography-Mass Spectrometry , Humans , Immunoblotting , Infant , Japan , Male , Metabolism, Inborn Errors/pathology , Tyrosine/bloodABSTRACT
Neonatal intrahepatic cholestasis is a heterogeneous disease of undetermined cause. There is an unreported subset of idiopathic neonatal intrahepatic cholestasis with an unusual histological combination of hepatic siderosis and macrovesicular steatosis. The patients were a 34-day-old female and a 39-day-old male with normal birth weights. Their mothers had received oral iron supplement 4-6 weeks before delivery. The patients had obstructive jaundice noticed at the well-baby clinic at 1 month of life. They had high levels of serum galactose and tyrosine, hyperferritinemia. Urinary organic acid and bile acid analyses were negative, and galactose-1-phosphate uridyltransferase activity in red cells was normal. Liver biopsies showed diffuse iron deposits and macrovesicular fat. By substituting formula milk with lactose-free milk, the patients responded, and had normal biochemical tests within 5 months of life. Follow-up biopsies, at the age of 12 months, showed mild residual fibrosis without iron or fat deposits. They are both well at 3 and 6 years of age, respectively, without biochemical liver dysfunction and neurologic impairment. Prenatal iron-overload might contribute to the pathogenesis of the disease, but further studies are needed to confirm the assumption.
Subject(s)
Cholestasis, Intrahepatic/complications , Fatty Liver/complications , Liver/metabolism , Siderosis/complications , Female , Humans , Infant, Newborn , MaleABSTRACT
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.
Subject(s)
17-Hydroxysteroid Dehydrogenases , Acetyl-CoA C-Acetyltransferase/metabolism , Bile Acids and Salts/biosynthesis , Enoyl-CoA Hydratase , Plant Proteins , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Animals , Bile Acids and Salts/metabolism , Caenorhabditis elegans/enzymology , Carrier Proteins/metabolism , Hydro-Lyases/metabolism , Isoenzymes/metabolism , Liver/enzymology , Multienzyme Complexes/metabolism , Peroxisomal Multifunctional Protein-2 , Rats , Sterols/metabolismABSTRACT
BACKGROUND/AIMS: Urinary 3-oxo-delta4 bile acids have been detected in infants who ultimately died of liver disease. We used qualitative and quantitative methods to compare urinary 3-oxo-delta4 bile acids in liver disease, determining their composition and evaluating the prognostic implication in patients of various ages with various liver diseases. METHODS: Gas chromatography-mass spectrometry was used to measure 3-oxo-delta4 bile acids in the urine of patients and healthy controls. RESULTS: Patients with a deficiency of 3-oxo-delta4-steroid 5beta-reductase and acute hepatic failure exhibited a significantly higher percentage of 3-oxo-delta4 bile acids in total bile acids in urine than the healthy controls or other patient groups, including those with neonatal cholestasis or biliary atresia (p<0.0001). The urinary 3-oxo-delta4 bile acids in patients with 3-oxo-delta4-steroid 5beta-reductase deficiency who had a poor prognosis were mainly 7alpha-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochola-4,6-dien-24-oic acid. CONCLUSIONS: Our results indicate that an increase in the 7alpha-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochola-4,6-dien-24-oic acid in the urine of patients with hepatobiliary disease indicates a poor prognosis.
Subject(s)
Biliary Atresia/urine , Chenodeoxycholic Acid/analogs & derivatives , Cholestasis/urine , Liver Diseases/urine , Oxidoreductases/deficiency , Adolescent , Adult , Aged , Biliary Atresia/complications , Case-Control Studies , Chenodeoxycholic Acid/urine , Child , Child, Preschool , Cholestasis/etiology , Female , Gas Chromatography-Mass Spectrometry , Humans , Infant , Liver Diseases/complications , Male , Middle AgedABSTRACT
The metabolism of bile acids in 30 pregnant women was evaluated by analyzing the urinary composition of bile acids during late gestation (weeks 30-41) and again in these women and their newborn infants during the first week after delivery. The levels of individual bile acids were determined by gas chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. The mean total bile acid/creatinine ratio in pregnant women decreased from 1.22 micromol/mmol creatinine at 30-32 weeks of gestation to 0.15 micromol/mmol creatinine at 6-7 days after delivery. The mean percentage of 1beta-hydroxylated bile acids peaked at 27% at 3-4 days after delivery. In newborn infants, the mean total bile acid/creatinine ratio rapidly increased from 3.39 micromol/mmol creatinine at birth to 54.33 micromol/mmol creatinine at 7 days. During this period, large amounts (40-50%) of unsaturated ketonic bile acids, especially 7alpha,12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholen-24-oic acid, were observed in the infants' urine. These data suggest that, during the perinatal period, the formation of polyhydroxylated and unsaturated ketonic bile acids probably represents a mechanism for the excretion of bile salts, and that the metabolism of bile acids in both the mother and the infant changes significantly after birth.
Subject(s)
Bile Acids and Salts/urine , Infant, Newborn/urine , Pregnancy/urine , Adult , Bile Acids and Salts/chemistry , Bile Acids and Salts/metabolism , Cholic Acids/urine , Creatinine/urine , Female , Gas Chromatography-Mass Spectrometry , Gestational Age , Humans , IsomerismABSTRACT
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.
Subject(s)
Bile Acids and Salts/metabolism , Liver/growth & development , Oxidoreductases/metabolism , Bile Acids and Salts/analysis , Bile Acids and Salts/urine , Creatinine/urine , Feces/chemistry , Female , Gas Chromatography-Mass Spectrometry , Humans , Infant , Infant, Newborn , Liver/enzymology , Male , Meconium/chemistry , Ursodeoxycholic Acid/urineABSTRACT
The mechanism of aromatic hydroxylation of estrogens by cytochrome P450 enzymes has been examined by comparing the oxidation of estrone with that of substrates carrying additional aromaticity such as equilenin and the structural analog 2-naphthol. Hamster liver microsomes preferentially catalyzed the conversion of estrone to 2-hydroxyestrone (Km = 30 and 25 microM and Vmax = 1497 and 900 pmol (mg of protein)-1 min-1 for 2- and 4-hydroxyestrone formation, respectively). In contrast, equilenin was hydroxylated exclusively at C-4 of the steroid ring system and 2-naphthol at the corresponding C-1 position (Km = 67 and 42 microM and Vmax = 2083 and 3226 pmol (mg of protein)-1 min-1 for 4-hydroxyequilenin and 1,2-dihydroxynaphthalene formation, respectively). This shift in the specificity of hydroxylation was due to the introduction of additional aromaticity at ring B of equilenin, because hamster liver microsomes are known not to contain any estrogen-4-hydroxylase, only estrogen-2-hydroxylase activity catalyzed by cytochrome P450 3A family enzymes. The exclusive 4-hydroxylation of equilenin is proposed to be due to a preferred delocalization of the naphthoxy radical an intermediate in the hydroxylation, to C-4, whereas delocalization to C-2 requires additional activation energy and is energetically not favored. Based on these electronic considerations, a mechanism of aromatic hydroxylation of estrogens is proposed which features hydrogen abstraction from the phenolic hydroxy group, electron delocalization of the phenoxy radical to a carbon-centered radical, and subsequent formation of catechol metabolites by hydroxy radical addition at C-2 or C-4 depending on steric or electronic constraints.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Estrogens/metabolism , Animals , Cricetinae , Equilenin/metabolism , Estrone/metabolism , Hydroxylation , In Vitro Techniques , Kinetics , Male , Mesocricetus , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , NADP/physiology , Naphthols/metabolism , Proadifen/pharmacologyABSTRACT
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.
Subject(s)
Bile Acids and Salts/biosynthesis , Hydroxyl Radical/metabolism , Liver/metabolism , Animals , Chromatography, Gas , Hydroxylation , Male , Oxidation-Reduction , Rats , Rats, Inbred WKY , Substrate SpecificityABSTRACT
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.
Subject(s)
Bile Acids and Salts/analysis , Chromatography, High Pressure Liquid/methods , Mitochondria, Liver/chemistry , Animals , Male , Molecular Structure , Rats , Rats, Wistar , Sensitivity and Specificity , Spectrometry, Fluorescence , StereoisomerismABSTRACT
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.
Subject(s)
Bilirubin/analysis , Pigmentation Disorders/pathology , Tooth Diseases/pathology , Animals , Disease Models, Animal , Female , Rats , Rats, Inbred StrainsABSTRACT
A method has been developed for the determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related bile acids in biological fluids of infants by gas chromatography-mass spectrometry (GC-MS) of the methyl ester-dimethylethylsilyl ether-methoxime derivatives. The 7alpha-hydroxylated 3-oxo-delta4-bile acids were partially dehydrated to give the 3-oxo-delta4,6-bile acids by trimethylsilyl or dimethylethylsilyl derivatization and other pretreatments under acidic or alkaline conditions for GC-MS analysis. To prevent dehydration, the 3-oxo-delta4-bile acids were derivatized to the oximes by treatment with O-methylhydroxylamine prior to pretreatments such as solid-phase extraction, enzymatic solvolysis and hydrolysis of the conjugates, and silylation with dimethylethylsilylimidazole. Calibration curves for the bile acids were linear over a range of 5-250 ng and the detection limit was 100 pg for each 3-oxo-delta4-bile acid. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free urine and serum ranged from 94.2 to 105.9% of their added amounts. The bile acids in urine and serum of four patients with severe cholestatic liver disease were measured by the analytical method, and the 3-oxo-delta4-bile acids were determined to be the major bile acids (59-68%) in the urines associated with 3-oxo-delta4-steroid 5beta-reductase deficiency or dysfunction.
Subject(s)
Bile Acids and Salts/blood , Bile Acids and Salts/urine , Cholestasis/blood , Cholestasis/urine , Gas Chromatography-Mass Spectrometry , Humans , InfantABSTRACT
The absolute configuration of 3 alpha,7 alpha,12 alpha, 24-tetrahydroxy-5 beta-cholestan-26-oic acid CoA ester (V-CoA) produced by the incubation of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid CoA ester (24E-THC-CoA) with D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-bifunctional protein) was investigated. When 24E-THC-CoA was incubated with D-bifunctional protein the formation of only one isomer (24R,25R-isomer) of four possible stereoisomers of V-CoA was observed, which suggested the cis-addition of water to a side chain double bond of 24E-THC-CoA. The dehydration reaction of V-CoA catalyzed by D-bifunctional protein was also observed when (24R,25R)-V-CoA was used as a substrate. The other three isomers (24R,25S-, 24S,25R- and 24S,25S-isomers) were not dehydrated with D-bifunctional protein. These results showed that D-bifunctional protein catalyzes stereospecifically the hydration and dehydration step in bile acid biosynthesis.
Subject(s)
17-Hydroxysteroid Dehydrogenases , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Cholic Acids/metabolism , Enoyl-CoA Hydratase/metabolism , Hydro-Lyases/metabolism , Multienzyme Complexes/metabolism , Saponins/metabolism , Triterpenes , Animals , Bile Acids and Salts/biosynthesis , Chromatography, High Pressure Liquid , In Vitro Techniques , Microsomes, Liver/enzymology , Peroxisomal Multifunctional Protein-2 , Proteins/metabolism , Rats , Stereoisomerism , Transferases/metabolismABSTRACT
A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1-100 pg and the detection limit for bile acids was 1 fg (S/N = 5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1 beta-, 2 beta-, 4 beta- and 6 alpha-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.