Enzymatic determination of cholesterol in bile

doi:10.1016/0009-8981(75)90364-2

Clinica Chimica Acta

Volume 64, Issue 3, 3 November 1975, Pages 337-341

https://doi.org/10.1016/0009-8981(75)90364-2 Get rights and content

Abstract

A simple, sensitive and rapid method for measuring the cholesterol concentration in bile is described. The method is based on the combination of an enzymatic technique and spectrophotometry. In the present study the results obtained with this method are compared with those obtained by the standard Liebermann-Burchard reaction on the same samples and are found to be in close agreement.

References (9)

J. Folch et al.
J. Biol. Chem.
(1957)
N. Kageyama
Clin. Chim. Acta
(1971)
C. Liebermann
Ber.
(1885)
L.A. Liese et al.
J. Biol. Chem.
(1962)

There are more references available in the full text version of this article.

Cited by (142)

The effects of L-carnitine-loaded solid lipid nanoparticles on performance, antioxidant parameters, and expression of genes associated with cholesterol metabolism in laying hens
2022, Poultry Science
Citation Excerpt :
Yolk cholesterol was estimated by the colorimetric Libermann-Burchard method at 560 nm (Boka et al., 2014) using the commercial kit. To determine the cholesterol concentration in bile, a simple, sensitive, and rapid method based on the combination of an enzymatic technique and spectrophotometry was used (Roda et al., 1975) by using a commercial cholesterol reagent kit. The serum GLU, TG, HDL, ALT, and AST values were determined using a Zhuoyue 310, Kehua Bio autoanalyzer (Shanghai, China), and the corresponding kits.
The purpose of this study was to investigate the production performance, antioxidant parameters, egg yolk cholesterol content, and expression of genes related to cholesterol metabolism in laying hens fed L-carnitine (LC) and L-carnitine-loaded solid lipid nanoparticles (LC-SLNs). A total of 350 Hy-Line (w-36) laying hens at 50 wk of age (1520.0 ± 0.7 g) were randomly assigned to 35 units (5 replicates and 50 hens in each treatment) with seven dietary treatments as a completely randomized design. The dietary treatments were corn-soybean meal-based diets, including 1) Control (basal diet); 2) Basal diet +50 mg/kg LC (50LC); 3) Basal diet +100 mg/kg LC (100LC); 4) Basal diet +150 mg/kg LC (150LC); 5) Basal diet +50 mg/kg LC-SLNs (50LC-SLNs); 6) Basal diet +100 mg/kg LC-SLNs (100LC-SLNs) and 7) Basal diet +150 mg/kg LC-SLNs (150LC-SLNs). Results showed that the 50LC-SLNs had the least feed conversion ratio (FCR) in all groups (P < 0.05). The dietary supplementation of 100LC-SLNs decreased (P < 0.01) the egg yolk cholesterol concentration from 14.71 to 11.76 mg/g yolk (25%). The 50LC-SLNs group produced the most total antioxidant capacity with a difference of 58.44% compared to the control group (P < 0.01). The greatest amount of total superoxide dismutase was found for 50LC-SLNs (P < 0.05), while the glutathione peroxidase was not affected by the experimental treatments (P > 0.05). Serum malondialdehyde levels were reduced by 50.52% in laying hens fed 50LC-SLNs compared to the control group (P < 0.05). The transcript level of 3-hydroxy-3-methylglutaryl coenzyme A reductase was significantly decreased (P < 0.01) in the LC and LC-SLNs groups. The expression of cholesterol 7α-hydroxylase was significantly increased (P < 0.01) in the plain LC (∼83%) and LC-SLNs (∼91%) groups. The inclusion of LC-SLNs in the diet increased (P < 0.05) the villus height and decreased villus width in all three parts of the small intestine. Dietary inclusion of LC was found to reduce egg yolk and serum cholesterol content by improving the production performance and antioxidant status. The LC-SLNs groups were more affected than the plain LC groups, which may be attributed to the increased bioavailability of LC.
Critical role of cholic acid for development of hypercholesterolemia and gallstones in diabetic mice
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
The composition of bile acids was determined by gas liquid chromatography as reported [12]. Biliary cholesterol and total bile acid concentration were quantitated by enzymatic methods [13,14], and phospholipids by the method described by Rouser et al. [15]. The relative concentrations of biliary lipids were expressed as molar percentages of the total biliary lipids.
We studied bile acid and cholesterol metabolism in insulin-dependent diabetes utilizing genetically modified mice unable to synthesize cholic acid (Cyp8b1−/−). Diabetes was induced in Cyp8b1−/− and wild type animals (Cyp8b1+/+) by alloxan, and the mice were fed normal or cholesterol-enriched diet for 10 weeks. The serum levels of cholesterol were strongly increased in diabetic Cyp8b1+/+ mice fed cholesterol, while diabetic Cyp8b1−/− mice did not show any aberrations regardless of the diet. Diabetic cholesterol-fed Cyp8b1+/+ mice had much higher biliary cholesterol and cholesterol saturation index than all other groups, their bile contained a large number of cholesterol crystals, and their canalicular cholesterol transporter Abcg5/g8 mRNA levels were much higher. Cyp7a1 mRNA levels were similar in all diabetic mice but higher compared to non-diabetic animals. The results indicate a critical role for cholic acid for the development of hypercholesterolemia and gallstones in our animal model.
Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice
2006, Journal of Lipid Research
Citation Excerpt :
The composition of bile acids in gallbladder bile was analyzed by gas-liquid chromatography, as reported (13). Total bile acid concentration was determined by an enzymatic method (14), as was cholesterol (15). Phospholipids were assayed by the method described by Rouser, Fleischer, and Yamamoto (16).
As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1^−/−) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1^+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1^−/− mice, compared to CYP8B1^+/+ mice. Cholesterol-fed CYP8B1^−/− mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1^+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.
Feedback regulation of bile acid synthesis in human liver: Importance of HNF-4α for regulation of CYP7A1
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
For the determination of the proportion between cholesterol and phospholipids bile was extracted with 20 volumes of chloroform:methanol (2:1, v/v). In the chloroform phase cholesterol was quantitated by an enzymatic method [9]. and phospholipids by spectrophotometric determination of phosphorous as described by Rouser et al. [10].
A great number of nuclear factors are involved in the negative feedback mechanism regulating bile acid synthesis. There are two major ways for the negative feedback to effect the synthesis; the SHP-dependent, involving FXR, and the SHP-independent way, affecting HNF-4α. We studied 23 patients with gallstone disease. Eight patients were treated with chenodeoxycholic acid, 7 with cholestyramine prior to operation, and 8 served as controls. Liver biopsies were analyzed with Real-time-PCR. In the cholestyramine-treated group mRNA levels of CYP7A1 were increased about 10-fold. Treatment with CDCA decreased the mRNA levels of CYP7A1 by about 70%. The mRNA levels of CYP8B1, CYP27A1, and CYP7B1 were not significantly altered in the treated groups. The analysis of mRNA levels for HNF-4α showed 64% higher levels in the cholestyramine-treated group compared to the controls. These levels showed positive and highly significant correlation to the levels of mRNA of CYP7A1 when studied in all three groups together. FXR, SHP, and LRH-1/FTF were not significantly affected by the different treatments. Our results indicate that when bile acid synthesis is upregulated by cholestyramine treatment the SHP-independent pathway for controlling CYP7A1 transcription dominates over the SHP-dependent pathway.
Sluggish small bowel motility is involved in determining increased biliary deoxycholic acid in cholesterol gallstone patients
1999, American Journal of Gastroenterology
OBJECTIVE: Our aim was to establish whether small intestine transit time is defective in subjects with cholesterol gallstones.
METHODS: We enrolled 10 patients (eight women, two men; mean age, 48.7 yr; mean body mass index [BMI], 22.4 Kg/m²) with recently diagnosed cholelithiasis, with no liver pathology, who were not taking any drugs, and 11 comparable healthy volunteers (eight women, three men; mean age, 46.2 yr; mean BMI, 22.7 Kg/m²), who served as controls. All subjects underwent orocecal (by starch breath test technique and serum assays of salazopyrin), oroileal (by serum assays of tauroursodeoxycholic acid), and duodenoileal (by serum assays of taurocholic acid) transit times; cholesterol saturation index; and bile acid composition and gallbladder motility studies (by ultrasound). For serum assays, blood samples were collected over a period of 7 h. Gallbladder motility and orocecal transit time were evaluated simultaneously.
RESULTS: All four means of assessing transit time gave longer times in cholesterol gallstone patients than in controls: orocecal transit time (salazopyrin) = 366 ± 13 vs 258 ± 16 min, p < 0.0005; orocecal transit time (starch breath test) = 415 ± 139 vs 290 ± 15 min, p < 0.01; duodenoileal transit time: 272 ± 23 vs 205 ± 23 min, p < 0.03; and oroileal transit time: 308 ± 18 vs 230 ± 19 min, p < 0.009. Cholesterol gallstone patients showed an increase in percent molar biliary deoxycholic acid (30% ± 4.5% vs 16% ± 1.3%, p < 0.02) and a decrease in percent molar cholic acid 32% ± 2.2% vs 40% ± 1.3%, p < 0.03) and chenodeoxycholic acid (34% ± 3% vs 41% ± 1.8%, p < 0.03), compared with controls; patients also had greater percent molar biliary cholesterol. A linear relationship (r² = 0.6324, p = 0.0012) between biliary deoxycholic acid and small bowel transit time was found. Residual gallbladder volumes were larger in cholesterol gallstone patients (11.38 ± 1.27 vs 7.55 ± 0.39 ml, p < 0.04), whereas basal gallbladder volumes, although higher, did not reach statistical significance (24.25 ± 2.41 vs 19.98 ± 1.63 ml; p = ns).
CONCLUSIONS: This study confirms that patients with cholesterol gallstones have delayed small bowel transit, defective gallbladder motor function, and increased biliary deoxycholic acid. Delayed small bowel transit may contribute to supersaturation of bile with cholesterol by increasing deoxycholic acid production.
Immobilization of cholesterol oxidase: An overview
2018, Open Biotechnology Journal

View all citing articles on Scopus

View full text

Brief technical noteEnzymatic determination of cholesterol in bile

Abstract

J. Biol. Chem.

Clin. Chim. Acta

Ber.

J. Biol. Chem.

Brief technical note
Enzymatic determination of cholesterol in bile