The diversity of ursodeoxycholic acid precursors from bile waste of commercially available fishes, poultry and livestock in Indonesia

Ursodeoxycholic acid (UDCA), a secondary bile acid (BA), has been used as a drug to treat various liver diseases. UDCA is synthesised from cholic or chenodeoxycholic acid (CA/CDCA), two primary BAs frequently used as the starting materials. Nowadays, swine, cattle, and poultry bile are the main sources of those BAs. However, other commercial animals could be promising sources as well. We identified two livestock, two poultries, and eight fishes that are commercially cultivated in Indonesia. Four free BAs including CA, CDCA, deoxycholic acid (DCA), and lithocholic acid (LA) were identified for their occurrences using thin-layer chromatography and high-performance liquid chromatography. CA was detected in cow, duck, red tilapia, gourami, the common carp, and grouper, whereas CDCA was only detected in two poultries and the common carp. The occurrence of DCA was common and abundant in most tested animals. In contrast, the presence of LA was found to be very low in all samples. The biliary bile of tilapia has been found to contain a high abundance of free CA (43% of the total bile). A simple extraction was able to purify CA from biliary bile of tilapia. This is a new promising and competitive source of CA.

Colestasis por deficiencia de 3β-Δ5-C27-hidroxiesteroide deshidrogenasa en un paciente con alteración en la síntesis de ácidos biliares / Cholestasis due to deficiency of 3β-Δ5-C27-hydroxysteroid dehydrogenase in a patient with altered bile acid synthesis

Resumen: Introducción: Los errores innatos en la síntesis de ácidos biliares son un grupo de defectos genéticos que representan del 1 al 2% de las enfermedades colestásicas crónicas en lactantes, niños y adolescentes. La deficiencia de 3β-Δ5-C27-hidroxiesteroide oxidoreductasa (3β-HSDH) es el defecto más comúnmente reportado. El cuadro clínico característico consiste en hepatitis neonatal, hepatomegalia, esplenomegalia, malabsorción, desnutrición y enfermedad hepática de aparición tardía. Caso clínico: Lactante masculino con antecedente de ictericia en escleras a los 4 meses que se resolvió espontáneamente; posteriormente, a los 18 meses, presentó enfermedad colestásica. Durante su abordaje se documentó gamma-glutamil transpeptidasa normal, hallazgo que es altamente sugestivo de alteración en la síntesis de ácidos biliares. El diagnóstico se realizó con espectrometría de masas en orina. Se inició tratamiento con ácido cólico oral, y presentó mejoría inmediata. Conclusiones: El resultado en los ácidos biliares urinarios es definitivo para el defecto genético y consistente con mutaciones homocigotas en el gen HSD3B7. Este padecimiento constituye un diagnóstico de exclusión en las enfermedades colestásicas de la infancia, particularmente el hallazgo de gamma-glutamil transpeptidasa normal o levemente aumentada, y responde adecuadamente al tratamiento oral, por lo que debe identificarse de forma temprana.

Abstract: Background: Inborn errors in bile acid synthesis are a group of genetic defects accounting for 1 to 2% of chronic cholestatic diseases in infants, children and adolescents. Deficiency of 3β-Δ5-C27-hydroxysteroid dehydrogenase (3β-HSDH) is the most common defect in this disease. Clinical features consist of neonatal hepatitis, hepatomegaly, splenomegaly, malabsorption, malnutrition, and late-onset liver disease. Case report: A male infant who presented jaundice in sclera at 4 months that resolved spontaneously, later presented cholestatic disease at 18 months. During his approach, normal gamma-glutamyl transpeptidase was documented, a finding that is highly suggestive of alteration in the synthesis of bile acids. The diagnosis was made using urine mass spectrometry. Oral colic acid treatment was started, presenting immediate improvement. Conclusions: The result in urinary bile acids is definitive for the genetic defect and consistent with homozygous mutations in the HSD3B7 gene. This condition is a diagnosis of exclusion in childhood cholestatic diseases, particularly in the presence of normal or mildly enlarged gamma-glutamyl transpeptidase, and responds adequately to oral treatment; it should be identified early.

An UPLC-MS/MS method for simultaneous determination of multiple constituents in Guizhi Fuling capsule with ultrafast positive/negative ionization switching / 中国天然药物

Guizhi Fuling capsule (GFC), a traditional Chinese medicine (TCM) with effects of promoting blood circulation and dissipating blood stasis, has been widely used in the clinic. Because of the complex matrix and various chemical structure types, quality control of GFC remains great challenge. In the present study, an ultra performance liquid chromatography hybrid triple-quadrupole mass spectrometry (UPLC-QQQ MS) method with ultrafast positive/negative ionization switching was developed for simultaneous determination of 18 bioactive components in GFC, including methyl gallate, ethyl gallate, oxypaeoniflorin, benzoic acid, albiflorin, paeonolide, paeoniflorin, 1, 2, 3, 4, 6-pentagalloylglucose, mudanpioside C, benzoyloxypaeoniflorin, benzoylpaeoniflorin, pachymic acid, amygdalin, cinnamaldehyde, paeonol, cinnamic acid, 4-hydroxybenzoic acid, and gallic acid. Separation was performed on an Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm), using a gradient elution with acetonitrile and water containing 0.1% formic acid. Cholic acid was selected as the internal standard. This newly developed method was fully validated for linearity, precision, accuracy, and stability, and then applied to quality assessment of GFC. Finally, the batch-to-batch reproducibility of GFC samples was evaluated by the cosine ration and Euclidean distance method, which showed high quality consistency. The results demonstrated that the developed method pro vided a reasonable and powerful manner for quality control of GFC.

An UPLC-MS/MS method for simultaneous determination of multiple constituents in Guizhi Fuling capsule with ultrafast positive/negative ionization switching / 中国天然药物

Guizhi Fuling capsule (GFC), a traditional Chinese medicine (TCM) with effects of promoting blood circulation and dissipating blood stasis, has been widely used in the clinic. Because of the complex matrix and various chemical structure types, quality control of GFC remains great challenge. In the present study, an ultra performance liquid chromatography hybrid triple-quadrupole mass spectrometry (UPLC-QQQ MS) method with ultrafast positive/negative ionization switching was developed for simultaneous determination of 18 bioactive components in GFC, including methyl gallate, ethyl gallate, oxypaeoniflorin, benzoic acid, albiflorin, paeonolide, paeoniflorin, 1, 2, 3, 4, 6-pentagalloylglucose, mudanpioside C, benzoyloxypaeoniflorin, benzoylpaeoniflorin, pachymic acid, amygdalin, cinnamaldehyde, paeonol, cinnamic acid, 4-hydroxybenzoic acid, and gallic acid. Separation was performed on an Agilent Zorbax Extend-C18 column (2.1 mm × 50 mm, 1.8 μm), using a gradient elution with acetonitrile and water containing 0.1% formic acid. Cholic acid was selected as the internal standard. This newly developed method was fully validated for linearity, precision, accuracy, and stability, and then applied to quality assessment of GFC. Finally, the batch-to-batch reproducibility of GFC samples was evaluated by the cosine ration and Euclidean distance method, which showed high quality consistency. The results demonstrated that the developed method pro vided a reasonable and powerful manner for quality control of GFC.

Effect of cholic acid on fetal cardiac myocytes in intrahepatic choliestasis of pregnancy / 华中科技大学学报（医学）（英德文版）

This study examined the effect of cholic acid (CA) on cultured cardiac myocytes (CMs) from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intrahepatic cholestasis of pregnancy (ICP). Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 μmol/L Ca(2+)-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.

Anti-inflammatory mechanism of qingfei xiaoyan wan studied with network pharmacology / 药学学报

This study aims to clarify out the anti-inflammatory mechanism of Qingfei Xiaoyan Wan. Chemical constituents of Qingfei Xiaoyan Wan identified by UPLC Q-TOF, were submit to Molinspiration, PharmMapper and KEGG bioinformatics softwares for predicting their absorption parameters, target proteins and related pathways respectively; and the gene chip and real time-PCR were carried out to investigate the expression of inflammatory genes on lung tissue of guinea pigs or human bronchial epithelial cell lines. The predicted results showed that 19 of the 24 absorbable constituents affected at 9 inflammation-related pathways through 11 protein targets; Qingfei Xiaoyan Wan treatment can significantly reduce the infiltration of cytokines through ERK1 gene and 5 inflammatory pathways (Focal adhesion, Fc epsilon RI, Toll-like receptors, NK cell-mediated cytotoxic, and ERK/MAPK). The results of real time-PCR further confirmed that the anti-inflammatory effects of Qingfei Xiaoyan Wan were due to active ingredients such as arctigenin, cholic acid and sinapic acid intervened focal adhesion, Fc epsilon RI signaling and ERK/MAPK pathways. The novel approach of 'drug-target-pathway' will present an effective strategy for the study of traditional Chinese medicines.

Bile Acid Inhibition of N-type Calcium Channel Currents from Sympathetic Ganglion Neurons

Under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted, regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases. Bile acids in circulation may affect the nervous system. We examined this possibility by studying the effects of bile acids on gating of neuronal (N)-type Ca2+ channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system. N-type Ca2+ channel currents were recorded from bullfrog sympathetic neuron under a cell-attached mode using 100 mM Ba2+ as a charge carrier. Cholic acid (CA, 10(-6) M) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution. CA suppressed the open probability of N-type Ca2+ channel, which appeared to be due to an increase in null (no activity) sweeps. For example, the proportion of null sweep in the presence of CA was ~40% at +40 mV as compared with ~8% in the control recorded without CA. Other single channel properties including slope conductance, single channel current amplitude, open and shut times were not significantly affected by CA being present. The results suggest that CA could modulate N-type Ca2+ channel gating at a concentration as low as 10(-6) M. Bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane. Under pathological conditions with increased circulating bile acids, CA suppression of N-type Ca2+ channel function may be beneficial against overexcitation of the synapses.

Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids / 药学学报

Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L.

Dietary chitosan enhances hepatic CYP7A1 activity and reduces plasma and liver cholesterol concentrations in diet-induced hypercholesterolemia in rats

The present study was performed to elucidate the hypocholesterolemic action of chitosan on the diet-induced hypercholesterolemia in rats. Male Sprague-Dawley rats (n=24) were fed with chitosan-free diet (Control), diets containing 2% or 5% chitosan for 4 weeks. Hypercholesterolemia was induced by adding 1% cholesterol and 0.5% cholic acid to all diets. Body weight gain and food intake of rats did not differ among the groups. The chitosan treated groups showed significant improvement in the plasma concentration of total cholesterol and LDL-cholesterol compared to the control group (p<0.05). Also, the chitosan treated groups decreased the liver concentration of total lipid and total cholesterol compared to the control group (p<0.05). The activity of hepatic cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids, was increased by 123% and 165% for the 2% or 5% chitosan diets, respectively. These findings suggest that enhancement of hepatic CYP7A1 activity may be a mechanism, which can partially account for the hypocholesterolemic effect of dietary chitosan in cholesterol metabolism.

Preparation and antitussive, expectorant and antiasthmatic activities of verticinone-bile acids salts / 药学学报

To search for potential drugs with potent antitussive, expectorant, antiasthmatic activities and low toxicity, a series of verticinone-bile acids salts were prepared based on the clearly elucidated antitussive, expectorant and antiasthmatic activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. The antitussive, expectorant and antiasthmatic activities of these verticinone-bile acid salts were then screened with different animal models. Ver-CA (verticinone-cholic acid salt) and Ver-CDCA (verticinone-chenodeoxycholic acid salt) showed much more potent activities than other compounds. The bioactivities of Ver-CA and Ver-CDCA are worthy to be intensively studied, and it is also deserved to pay much attention to their much more potent antitussive effects than codeine phosphate. In order to elucidate whether they have synergistic effect and attenuated toxicity, their activities will be continuously compared with single verticinone, cholic acid and chenodeoxycholic acid at the same doses on different animal models. The application of "combination principles" in traditional Chinese medicinal formulations may be a novel way in triditional Chinese medicine research and discovery.

Effects of Corn Peptide Consumption on Plasma Lipid Profiles in Cholesterol-Fed Rats

In this study, the effects of corn peptide consumption on plasma lipid profiles were investigated in high cholesterol dietfed rats. Male Sprague-Dawley rats (n = 21) were fed with corn peptide-free (control) diet, diets containing 2% or 5% corn peptide for 5 weeks. Hypercholesterolemia was induced by adding 1% cholesterol and 0.5% cholic acid to all diets. No difference was found in food intake and body weight gain among groups. The corn peptide treated groups showed significant improvement in the plasma level of HDL-cholesterol (p < 0.05) compared to the control group, while the plasma total cholesterol and LDL-cholesterol were not affected. 5% corn peptide supplemented diet reduced plasma level of triglycerides (p < 0.05). The atherogenic index was decreased in the corn peptide treated groups. These results suggest that consumption of corn peptide may lead to an amelioration of metabolic syndrome as well as a reduction of cardiovascular disease and hyperlipidemia through increasing the level of HDL-cholesterol, and decreasing the level of triglycerides in plasma.

Effect of Korean Red Ginseng on Vaginal Blood Flow and Structure in Hypercholesterolemic Female Rats / 대한남성과학회지

PURPOSE: The aims of this study were to investigate the effects of Korean Red Ginseng(KRG) on vaginal blood flow and histological changes in a hypercholesterolemic female rat model. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into two groups: the control(n=20) and the hypercholesterolemia(n=40). Hypercholesterolemia group was fed a high fat diet(2% cholesterol, 1% cholic acid, 5% coconut oil) for 12 weeks. The hypercholesterolemia group was further divided into the vehicle only and the KRG treatment(50 mg/kg/day) groups. After 4 and 8 weeks of treatment, vaginal blood flow was measured by a laser Doppler flowmeter. Vaginal tissues were processed for histology and Western blot. RESULTS: After 4 and 8 weeks of treatment, serum cholesterol levels(mg/dl) were significantly increased in the hypercholesterolemia group(1185.0+/-736.1, 934.3+/-212.3) compared with the control group(69.7+/-19.5, 67.1+/-7.2), and partially decreased in KRG treatment group(688.2+/-251.5, 694.2+/-150.4), respectively. Vaginal blood flow(ml/min/100 g tissue) after pelvic nerve stimulation was lower in the hypercholesterolemia group(17.3+/-7.9, 17.9+/-5.5) compared with the control group(27.3+/-17.1, 26.9+/-16.4), however, the KRG treatment group(29.5+/-10.3, 27.4+/-11.1) was as high as the control group, respectively. The expressions of TGF-beta1 tended to increase in the vagina of the hypercholesterolemia animals compared to those of the control and the KRG treatment groups. CONCLUSIONS: KRG treatment in hypercholesterolemic female rats decreased serum cholesterol levels and improved vaginal blood flow. These results suggest that KRG treatment may have a beneficial effect in women's sexual health.

The protective effect of cholic acid on an in vitro injury model of ischemia-reperfusion in cerebral microvascular endothelial cells / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an in vitro injury model of ischemia-reperfusion in cerebral microvascular endothelial cells of rats and observe the protective effect of cholic acid.</p><p><b>METHOD</b>Cultured rat microvascular endothelial cells were subjected to the oxygen-glucose deprivation (OGD) (Krebs solution) and recovery of oxygen-glucose, which simulated in vitro ischemia and reperfusion injury, and treated with cholic acid. The A value was measured with MIT chromatometry.</p><p><b>RESULT</b>Cultured cells were impaired after OGD for 4 hours and recovery of oxygen-glucose for 12 hours, the A value of the cells treated with cholic acid was significantly higher than that of the cells without treatment (P < 0.01).</p><p><b>CONCLUSION</b>Cholic acid could obviously protect rat cerebral microvascular endothelial cells from injury induced by an in vitro ischemia-reperfusion.</p>

Extraction of cholic acid from animal bile and semi-synthesis of chenodexy cholic acid

From the cow's bile, which is by product of food manufacture installations in Ho Chi Minh city, was surveyed and built up a progress for isolating cholic acide. From cholic acide by five steps obtained chenodeoxycholic acide. The product come up to the standard of BP 2000

Alterações hepatobiliares induzidas pelos ácidos cólico e litocólico: estudo experimental em hamsters / Hepatobiliaries alterations induced by cholic and lithocholic acids: hamsters experimental study

A atresia de vias biliares é a causa mais comum de icterícia obstrutiva, cirrose e transplante hepático da infância. Sua etiopatogenia permanente desconhecida. Dentre várias teorias, uma propõe que a enfermidade pode ser causada pelo efeito tóxico de ácidos biliares monohidroxilados no sistema hepatobiliar fetal e neonatal. As características do metabolismo biliar nesta fase da vida e possíveis alterações bioquímicas desses ácidos poderiam causar reaçäo inflamatória e obstruçäo ductal. Ainda näo foi feito qualquer estudo experimental da açäo desses ácidos sobre o sistema hepatobiliar durante a gravidez. Neste trabalho, avaliaram-se os efeitos tóxicos provocados pela ingestäo de um ácido biliar trihidroxilado, o cólico, e um monohidroxilado, o litocólico, sobre o sistema hepatobiliar de hamsters durante os períodos gestacional e perinatal. A escolha deste animal deve-se à semelhança de seu metabolismo biliar com o humano. A ingestäo de ácido litocólico a 0,5 por cento durante a gestaçäo de hamsters, provocou proliferaçäo ductal/ductular acentuada, sinais inflamatórios, degeneraçäo e regeneraçäo celular hepática, hiperplasia do epitélio dos ductos extra-hepáticos maternos e aborto. Tanto o ácido cólico a 0,5 por cento como o ácido litocólico a 0,25 por cento, quando ingeridos por hamsters grávidas, provocaram proliferaçäo ductal/ductular e lesäo inflamatória hepatobiliar em graus variáveis no animal adulto e de leve intensidade nos filhotes. Induziu, ainda, a reduçäo da ninhada. Portanto, verificou-se que a ingestäo destes ácidos biliares por hamsters durante o período gestacional provocou toxicidade variável sobre o sistema hepatobibliar materno e de recém-nascidos

Gastric Stump Cancer

PURPOSE: Gastric stump cancer is defined as a cancer that develops in the stomach after a resection in cases of non-malignant or malignant gastric disease. The interval between the gastrectomy and the detection of gastric stump cancer must be over 5 years. Since duodenogastric reflux gastritis is a precancerous condition and one of the most important factors inducing gastric stump cancer, we compared the bile-acid content of gastric juice between gastric stump cancer patients and controls. MATENRIALS AND METHODS: To evaluate retrospectively the surgical treatment of patients with gastric stump cancer, we reviewed the cases histories of 1016 stomach cancer patients who had been operated on at the Department of General Surgery, Kosin University Gospel Hospital, between 1995 and 1998. The gastric juice was collected during the operations on the gastric stump cancer patients by using a needle puncture of the fundus of the stomach and during the endoscopic examinations of the control subjects. The samples were analyzed for various bile acids (gas chromatography/mass spectrometry). RESULTS: The 6 gastric stump cancer cases accounted for 0.6% of all gastric cancer patients; 5 patients were first operated on for a peptic ulcer and the remaining one for an adenocarcinoma of the stomach. All of the cases were men. The reconstruction method after the initial gastrectomy was a Billroth II in all cases. The sites of the gastric stump cancer were the anastomotic sitein 2 patients, the upper body in 2, the fundus in 1 and the cardia in 1. The operative methods were 3 total gastrectomies, 2 subtotal gastrectomies with Roux en Y anastomosis, and 1 partial gastrectomy with lymph node dissection and had a curative intention in all patients. All of the patients were still surviving at the time of this report. The gastric juices of 4 gastric stump patients showed significantly higher contents of cholic acid (36.42microgram/ ml) compared to the gastric juices of 35 control subjects (12.82microgram/ml)(p< or =0.0001). Chenodeoxycholic acid and lithocholic acid were not significantly different. CONCLUSION: The gastric juice of gastric stump cancer patients contained a significantly higher cholic acid content. At the time of the initial gastrectomy, an operative method that prevents duodenogastric reflux may prevent or minimize the development of gastric stump cancer, and more aggressive surgical treatment may improve survival.

Bile acid modulation of gastroinstinal smooth muscle contraction and ionic currents

We have examined whether bile acids can affect the electrical and mechanical activities of circular smooth muscle of canine colon and ileum, using isometric tension measurement or patch clamp technique. It was found that a dilution of canine bile (0.03 ~ 2% by volume) enhanced or inhibited the amplitude of spontaneous contractions. An individual component of bile, deoxycholic acid (DCA) enhanced the frequency and amplitude of the spontaneous contractile activity at 10(-6) M, while DCA at 10(-4) M inhibited the contraction. Similarly, the response to cholic acid was excitatory at 10(-5) M and inhibitory at 3 X 10(-4) M. Taurocholic acid at 10(-4) M enhanced the amplitude of muscle contraction. Electrically, canine bile at 1% reversibly depolarized the colonic myocytes under current clamp mode. Bile acids also elicited non-selective cation currents under voltage clamp studies, where K+ currents were blocked and the Cl- gradient was adjusted so that ECl- was equal to -70 mV, a holding potential. The non-selective cation current might explain the depolarization caused by bile acids in intact muscles. Furthermore, the bile acid regulation of electrical and mechanical activities of intestinal smooth muscle may explain some of the pathophysiological conditions accompanying defects in bile reabsorption.

Electron Misoscopic Morphology of the Gallbladder and Gallstone Formation induced by High Cholesterol-Cholic Acid Diet in Mice / 대한간학회지

BACKGROUND/AIMS: The experimental production of cholesterol gallstone suggests that alteration of the nature of gallbladder mucus may be a factor in the induction of gallbladder stones. This study was designed to clarify the changes of the gallbladder epithelia and the role of a high cholesterol-cholic acid diet (CCD) in the formation of the gallstones. METHOD: Forty mice were fed on a diet of natural 1% high-cholesterol egg yolk and 2.5% cholic acid for 8 weeks. The mice were sacrificed biweekly and followed up for 8 weeks. The gallbladder tissues and bile were prepared for electron microscopic examination. We observed the ultrastructural changes of the epithelia and the sediments of the gallbladder bile with scanning and transmission electron microscopic observation. RESULTS: 1) Process of Gallstone formation in bile: At the 2nd week in mice fed with CCD, the crystals grew up by appositional layering into monoliths and then by aggregation into mulberry-shaped stones. At the 8th week many stones and numerous concrements were observed. 2) Scanning electron microscopic findings: In mice fed with CCD, the scattered whitish patches of epithelial cell surfaces exhibited the cauliflower-like apical protrusion secreting mucin. These protrusions appeared to be swellings of the apical plasmalemma and they were mixed with the mucus and microvilli. 3) Transmission electron microscope findings: At the 2nd week, numerous electron lucent secretory granules were observed in the apical zone of the epithelial cells. Increased numbers of fat vacuoles were also observed in the basal area of epithelium. Two types of cells were observed in the lining epithelium: dark and light cells. CONCLUSION: This study suggests that long-term administration of CCD, and the resultant increased secretion of glycoprotein from gallbladder epithelial cells, could be a factor in the induction of the gallstones.

Gallstone formation and gallbladder mucosal changes in mice fed a lithogenic diet

To investigate the pathologic change of gallbladder mucosa related to gallstone formation, 52 mice were fed a lithogenic diet containing 1% cholesterol and 0.5% cholic acid and we evaluated the sequential morphologic changes in the gallbladder from two days to 40 weeks. Cholesterol gallstones began to appear after two weeks and all the mice had gallstones after eight weeks. At two days, the mitotic index was at its highest. The gallbladder mucosa showed progressive hyperplastic change with earlier papillary projection of the folds and later inward proliferation. At the same time of stone formation, mucous cells forming glands appeared. Their histochemical profile of mucin was different from that of normal epithelium. Numbers of mucous cells increased gradually until 24 weeks but slightly decreased afterward. These results suggest hyperplasia and metaplasia are closely related to the gallstone formation. Hyperplasia is probably reactive to irritating effect of lithogenic bile or stone. Metaplasia and cholesterol gallstone may develop simultaneously, and act synergistically.