Changes in in vivo metabolism of bile acids in rat after treatment with phenobarbital

The influence of phenobarbital on pool size and turnover of bile acids in rats have been investigated by administration of [24-¹⁴C] cholic acid and tritium labeled chenodeoxycholic acid. Phenobarbital treated rats had a smaller cholic acid pool compared to control rats (6.08±2.09 mg and 23.60±7.66 mg, respectively). The pool size of chenodeoxycholic acid, plus its metabolites (α- and β-muricholic acids), was of the same magnitude in the two groups of animals. Also the daily production of cholic acid was decreased in phenobarbital treated rats compared to control rats (2.12±0.46 mg and 7.24±1.66 mg, respectively). No significant difference was observed between the synthesis of chenodeoxycholic acid in the two groups of animals.     

Evidence for distinct precursor pools for biliary cholesterol and primary bile acids in cebus and cynomolgus monkeys

The abnormal metabolism and distribution of plasma lipoproteins have been associated with atherosclerosis and gallstones. To better understand the process of cholesterol excretion, a study was designed to determine whether the contribution of lipoprotein free¹⁴C-cholesterol (as LDL or HDL) to biliary cholesterol or primary bile acids differs in two species of nonhuman primates, cebus and cynomolgus monkeys, having opposite plasma LDL/HDL ratios. Since amino acid conjugation might influence bile acid synthesis or secretion, the taurine and glycine conjugates of newly synthesized primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), were measured in the species capable of conjugating with taurine or glycine (cynomolgus). After total bile acid pool washout, monkeys were infused with human LDL or HDL labeled with free¹⁴C-cholesterol, and the specific activities (SA) of biliary cholesterol and primary bile acid conjugates were determined. In both species, regardless of the lipoprotein infused, the SA of biliary cholesterol and CA were greater than those for total bile acids and CDCA, respectively. In cynomolgus, the SA of glycine conjugates was higher for CA than CDCA, while the SA of taurine conjugates was greater for CDCA than CA. Under these conditions, (i) infused lipoprotein free cholesterol (as either LDL or HDL) contributed more to biliary cholesterol than to bile acids and more to CA than to CDCA; (ii) glycine conjugated preferentially with CA rather than CDCA, while taurine was the preferred conjugate for CDCA. Further, whereas the two primary bile acids had similar rates of synthesis and turnover in cynomolgus, basal bile acid synthesis was much greater in cebus and the CDCA turnover appeared disproportionately large.     

Glucagon and N6,O2′-dibutyryl adenosine 3′∶5′-monophosphate inhibition of lipogenesis and phosphofructokinase activity of hepatocytes from meal-fed rats

Glucagon and N⁶,O^2′-dibutyryl adenosine 3′∶5′-monophosphate (dibutyryl cyclic AMP) inhibit net glucose utilization, lactate plus pyruvate accumulation and fatty acid synthesis by isolated hepatocytes prepared from meal-fed rats. A crossover in the metabolite profile of the glycolytic intermediates occurs between fructose-6-phosphate and fructose-1,6-bisphosphate, suggesting either inhibition of phosphofructokinase or activation of fructose diphosphatase, or both. Direct assay of the enzymes in cell-free extracts of the hepatocytes indicates that dibutyryl cyclic AMP inhibits phosphofructokinase but has no effect upon fructose diphosphatase. The assay for phosphofructokinase was modified by the use of ITP in place of ATP for the phosphate donor as the ATP-linked assay is complicated by an apparent time-dependent activation of the enzyme. These findings strongly suggest that cyclic AMP inhibition of phosphofructokinase explains in part cyclic AMP inhibition of aerobic glycolysis and lipogenesis by rat liver hepatocytes.     

Biosynthesis of fatty acids byTrypanosoma cruzi

The incorporation of [1-¹⁴C] acetate into fatty acids by cultured epimastigotes ofTrypanosoma cruzi was studied. After 8, 24, and 48 hr incubation with labeled precursor, up to 2.8% of the initial radioactivity added to the medium was found in theT. cruzi long chain fatty acids. Saturated (16∶0 and 18∶0), monounsaturated (18∶1ω9), and diunsaturated (18∶2ω6) fatty acids were synthesized. Both the pattern of incorporation of labeled acetate into the fatty acids and the decarboxylation ratios found suggest that de novo synthesis of fatty acids has taken place.     

Stearic acid unlike shorter-chain saturated fatty acids is poorly utilized for triacylglycerol synthesis and β-oxidation in cultured rat hepatocytes

Utilization of stearate as compared to various saturated fatty acids for cholesterol and lipid synthesis and β-oxidation was determined in primary culture of rat hepatocytes. At 0.5 mmol/L in the medium, stearate (18:0) adequately solubilized by albumin was less inhibitory to cholesterol synthesis from [2-¹⁴C] acetate than myristate (14:0) and palmitate (16:0) (68% vs. 91 and 88% inhibition, respectively). The rate of incorporation into cholesterol from [1-¹⁴C] stearate (3.0±0.6 nmol/mg protein/4 h) was 37-, 1.8-, and 7.8-fold of that from myristate, palmitate, and oleate, respectively. Conversely, the rate of [1-¹⁴C] stearate incorporation into total glycerolipids was 88–90% lower than that of labeled palmitate, myristate, and oleate. The rate of [1-¹⁴C] stearate incorporation into triacylglycerol (3.6±0.4 nmol/mg protein/4 h) was 6–8% of that from myristate, palmitate, oleate, and linoleate. The rate of stearate incorporation into phospholipids was the lowest among tested fatty acids, whereas the rate of mono- and diacylglycerol synthesis was the highest with stearate treatment. The rate of β-oxidation as measured by CO₂ and acid soluble metabolite production was also the lowest with [1-¹⁴C] stearate treatment at 22.7 nmol/mg protein/4 h, which was 35–40% of those from other [1-¹⁴C] labeled fatty acids. A greater proportion of stearate than other fatty acids taken up by the hepatocytes remained free and was not metabolized. Clearly, stearate as compared to shorter-chain saturated fatty acids was less efficiently oxidized and esterified to triacylglycerol in cultured rat hepatocytes.     

Comparative effect of glucagon,dibutyryl cyclic AMP,and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes

The effect of glucagon, dibutyryl cyclic adenosine 3′,5′-monophosphate, and epinephrine on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied. The incubations were performed with rat liver microsomes and labeled linoleic acid under desaturating and elongating conditions. Under desaturating conditions, linoleic acid was converted to γ-linolenic acid, whereas under elongating conditions it was converted to 20∶2ω6. Glucagon, dibutyryl cyclic AMP, and epinephrine decreased the oxidative desaturation of linoleic acid to γ-linolenic acid while the elongating reaction was not modified in the experimental conditions tested. Consequently, the results support the hypothesis that the oxidative desaturation of linoleic acid to γ-linolenic acid is the main controllable step in the biosynthesis of polyunsaturated fatty acids of the linoleic acid family in the microsomes.     

Incorporation and distribution of saturated and unsaturated fatty acids into nuclear lipids of hepatic cells

Liver nuclear incorporation of stearic (18∶0), linoleic (18∶2n−6), and arachidonic (20∶4n−6) acids was studied by incubation in vitro of the [1-¹⁴C] fatty acids with nuclei, with or without the cytosol fraction at different times. The [1-¹⁴C] fatty acids were incorporated into the nuclei as free fatty acids in the following order: 18∶0>20∶4n−6≫18∶2n−6, and esterified into nuclear lipids by an acyl-CoA pathway. All [1-¹⁴C] fatty acids were esterified mainly to phospholipids and triacylglycerols and in a minor proportion to diacylglycerols. Only [1-¹⁴C] 18∶2n−6-CoA was incorporated into cholesterol esters. The incorporation was not modified by cytosol addition. The incorporation of 20∶4n−6 into nuclear phosphatidylcholine (PC) pools was also studied by incubation of liver nuclei in vitro with [1-¹⁴C]20∶4n−6-CoA, and nuclear labeled PC molecular species were determined. From the 15 PC nuclear molecular species determined, five were labeled with [1-¹⁴C]20∶4n−6-CoA: 18∶0–20∶4, 16∶0–20∶4, 18∶1–20∶4, 18∶2–20∶4, and 20∶4–20∶4. The highest specific radioactivity was found in 20∶4–20∶4 PC, which is a minor species. In conclusion, liver cell nuclei possess the necessary enzymes to incorporate exogenous saturated and unsaturated fatty acids into lipids by an acyl-CoA pathway, showing specificity for each fatty acid. Liver cell nuclei also utilize exogenous 20∶4n−6-CoA to synthesize the major molecular species of PC with 20∶4n−6 at the sn-2 position. However, the most actively synthesized is 20∶4–20∶4 PC, which is a quantitatively minor component. The labeling pattern of 20∶4–20∶4 PC would indicate that this molecular species is synthesized mainly by the de novo pathway.     

Effect of bile acid feeding on hepatic steroid 12 α-hydroxylase activity in hamsters

The effects of feeding cholic acid, chenodeoxycholic acid and ursodeoxycholic acid on the activity of the hepatic steroid 12α-hydroxylase, gallbladder bile acid composition, fecal neutral sterol output, cholesterol synthesis and bile acid synthesis were determined in female hamsters. The 12α-hydroxylase activity was inhibited to 56% by cholic acid, to 62% by chenodeoxycholic acid, and to 78% by ursodeoxycholic acid compared with the control. Bile acid composition was altered by feeding of cholic acid and chenodeoxycholic acid to be rich in the given bile acids. Fecal neutral sterol output increased about twice by feeding chenodeoxycholic acid and ursodeoxycholic acid, whereas cholic acid had no significant effect. Body cholesterol synthesis increased to 217% by chenodeoxycholic acid and to 274% by ursodeoxycholic acid, whereas effect of cholic acid was not significant. Bile acid synthesis was suppressed to 48% of control only by chenodeoxycholic acid. A positive correlation between the 12α-hydroxylase activity and the bile acid synthesis was observed in the control, chenodeoxycholatefed and ursodeoxycholate-fed animals. In conclusion, ursodeoxycholic acid might have less inhibitory effect on the steroid 12α-hydroxylase and the bile acid synthesis than chenodeoxycholic acid.     

Enterohepatic circulation of cholic acid and cholesterol metabolism in the rat

A role for cholic acid in the regulation of body cholesterol levels is emphasized by the following experiment and findings. Fifteen rats with biliary and duodenal fistulae were divided into three groups. 4-¹⁴C-cholesterol was admistered intravenously to all animals on day 1. Two groups received a duodenal infusion of either sodium taurocholate or of cholesterol at the same time and thereafter daily. Bile samples were collected daily for 15 consecutive days. Cholic, chenode-oxycholic acid and cholesterol were the major labeled compounds in the bile. Labeled cholic acid disappeared from the bile of control animals after day 8 while it persisted in the group receiving unlabeled cholic acid up to day 15. The decrease of specific radioactivity of labeled biliary cholic acid in the rats receiving unlabeled cholesterol corresponded to that of control animals. A significant increase in the concentration of cholesterol was found in the plasma of animals receiving cholic acid and in the liver of those receiving unlabeled cholesterol. A preliminary report was presented at the 49th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, 1965.     

Biosynthesis of fatty acids in cell-free homogenates of lactating gerbil mammary gland

Cell-free homogenates of lactating mammary gland of gerbils maintained on a diet of sunflower seed, guinea pig chow and oats (Diet 1) or a diet of guinea pig chow and oats (Diet 2) and of rats maintained on laboratory chow (Diet 3) were incubated with¹⁴C-labeled acetate, acetyl CoA or malonyl CoA aerobically. A large proportion of the¹⁴C from¹⁴C-acetate and¹⁴C-acetyl CoA incorporated into fatty acids by homogenates from gerbils on Diet 1 was in unsaturated compounds, particularly in 18-carbon and 20-carbon dienoic acids, compared to preparations from animals on Diets 2 or 3. The two radioactive dienoic acids were proven to be Δ^9,12 18∶2 and Δ^11,14 20∶2, and the latter was shown to be a direct elongation product of Δ^9,12 18∶2 by the substrate¹⁴C-acetyl CoA. In all experiments¹⁴C from¹⁴C-malonyl CoA was incorporated predominantly into 14∶0 and 16∶0, and very little incorporation occurred into unsaturated fatty acids in homogenates made either from gerbil or rat. Total fatty acids isolated from homogenates and from milk fat (fat floating on the centrifuged homogenates) of gerbils on Diet 1 had a higher proportion of 18∶2 than animals on the other two diets, a reflection of the large dietary intake of linoleic acid by gerbils on Diet 1. Under these conditions the amount of 18∶2 in the mammary gland had a significant effect on the products of the incubation.     

Effects of feeding chenodeoxycholic acid on metabolism of cholesterol and bile acids in germ-free rats

The aim of this investigation was to study the influence of chenodeoxycholic acid administration on cholesterol and bile acid synthesis in germ-free rats. Seven rats were fed a basal diet and 2 groups of 4 rats received the same diet supplemented with 0.4 and 1% chenodeoxycholic acid, respectively. After 6 weeks, feces were collected in one 3- and one 4-day pool for analysis of cholesterol and bile acids. When the sampling period was finished, the rats were killed and the liver microsomal fractions isolated. The activities of HMG CoA reductase and cholesterol 7α-hydroxylase were determined, the 7α-hydroxylase by a mass fragmentographic method. The 2 dominating bile acids in the untreated rats were cholic acid and β-muricholic acid. During treatment with chenodeoxycholic acid, 60–70% of this bile acid was converted into α- and β-muricholic acid, indicating a high activity of the 6β-hydroxylase. The excretion of cholic acid was almost completely inhibited and the 7α-hydroxylase activity was decreased ca 75% in the rats fed 1% chenodeoxycholic acid. The activity of the hepatic HMG CoA reductase was unchanged. The fecal excretion of cholesterol increased 2–3 times. An accumulation of cholesterol was seen in the rats treated with 1% chenodeoxycholic acid, which was probably a result of the decreased catabolism of cholesterol to bile acids.     

Biosynthesis of [14C]arachidonic acid from [14C]linoleate in primary cultures of rat Sertoli cells

The conversion of [¹⁴C]linoleate to [¹⁴C]arachidonate by rat Sertoli cells was established by use of primary cultures. Most of the¹⁴C from [1-¹⁴C]linoleate was located in C-3 of the synthesized arachidonate, indicating that the labeled tetraene had originated largely by elongation and desaturation of the intact labeled substrate rather than by mere addition of¹⁴C-acetate generated by bio-oxidation of the radioactive substrate to an already existing 18-carbon precursor. Although a relatively small amount of¹⁴C was present in 18∶3ω6 and a relatively large amount of¹⁴C was present in 20∶2, it was not possible from these data to establish the relative importance of 20∶2 in the biosynthesis of arachidonic acid in rat Sertoli cells.     

Absorption and biliary secretion of intraperitoneally injected 3β-methoxycholest-5-ene-4-14C in the rat

Following ip injection of 1 mg amounts of methoxycholestene-4-¹⁴C into normal intact rats, the amount of label in the whole liver was measured at different time intervals in different animals. These studies indicated that most of the injected compound was absorbed by or before 12 hr. In bile duct-cannulated rats the secretion of label from ip injected methoxycholestene-4-¹⁴C or cholesterol-4-¹⁴C into bile was usually less during the first 24 hr than during the second 24 hr after injection. Studies on the nature of the labeled compounds in the bile following ip injection of methoxycholestene-4-¹⁴C indicate that most of the label is present in metabolites, 47% of the label in cholic acid and 31% in chenodeoxycholic acid.     

Hypolipidemic effects of β-cyclodextrin in the hamster and in the genetically hypercholesterolemic rico rat

The effect of increasing amounts of a cyclic oligosaccharide, β-cyclodextrin (BCD), included in the diet on plasma cholesterol and triglycerides, was investigated in two animal models, namely in male genetically hypercholesterolemic Rico rats and in male Syrian hamsters. The distribution of bile acids in the gastrointestinal tract and in the feces of hamsters was also determined. In the Rico rats and hamsters, plasma cholesterol and triglycerides decreased linearly with increasing doses of BCD. In these two species, 20% BCD as compared to control diet lowered cholesterolemia (−35%) and triglyceridemia (−70%). In the hamster, the BCD diet caused a marked decrease in cholesterol and triglycerides in chylomicrons and very low density lipoprotein, and in high density lipoproteins cholesterol. Composition and amounts of bile acids were modified in the gastrointestinal tract of hamsters receiving 10% BCD as compared to the control group. The total bile acid content of the gallbladder of treated hamsters was fourfold higher than in the control group, and the bile contained a large amount of hydrophilic bile acids. This trend was also observed in the small intestine, in which percentages and total quantities of cholic plus deoxycholic acids (cholic pathway) were higher than those of chenodeoxycholic plus ursodeoxycholic plus lithocholic acids (chenodeoxycholic pathway). The bile acid contents of the cecum and colon of treated hamsters were 2.7-fold higher than those of control animals, but the bile acid composition was similar in the two groups of hamsters. Fecal excretion of bile acids was 3.3-fold higher in the treated group than in the control group, and the percentage of lithocholic acid was markedly increased and close to that observed in the colon. The turnover of the chenodeoxycholic pool was twice as fast in treated hamsters as in control hamsters, whereas that of cholic acid was not significantly modified. These results suggest that BCD does not alter the microbial degradation of bile acids, but rather stimulates their synthesis and increases their pool size. BCD prevents the intestinal absorption of lithocholic acid and washes this cytotoxic bile acid from the colon. The hypocholesterolemic effect of BCD appears to be due to stimulation of bile acid synthesis.     

Pathways for fatty acid elongation and desaturation in Neurospora crassa

Neurospora crassa incorporated exogenous deuterated palmitate (16∶0) and ¹⁴C-labeled oleate (18∶1^Δ9) into cell lipids. Of the exogenous 18∶1^Δ9 incorporated, 59% was desaturated to 18∶2^Δ9,12 and 18∶3^Δ9,12,15. Of the exogenous 16∶0 incorporated, 20% was elongated to 18∶0, while 37% was elongated and desaturated into 18∶1^Δ9, 18∶2^Δ9,12, and 18∶3^Δ9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18∶0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18∶0, indicating a sixfold preferential use of 16∶0 for saturated fatty acid synthesis. These results indicate that the release of 16∶0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.     

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 10¹⁰ counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7α-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-β-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an imporant role in the formation of secondary bile acids and coprostanol in rats.     

Hepatic bile acid elution by albumin and bile acid content in isolated rat hepatocytes

Bile acid contents were determined for isolated rat hepatocytes. During the course of isolating the hepatocytes, perfusion of rat liver with buffer containing 2% albumin eluted a significant amount of bile acids. The elution was proportional to the volume of the buffer and attributable to albumin in the buffer. The isolated hepatocytes prepared by perfusion with 0.1% albumin buffer, which eluted a negligible amount of bile acids, contained 95±12 μg/10⁸ cells of bile acids. The major bile acids were cholic acid (22%), β-muricholic acid (34%) and hyodeoxycholic acid (10%). Levels of the other bile acids were less than 3%. Peak 8, unidentified but presumed to be a trihydroxycholanoic acid, accounted for 19%.     

Absorption and metabolic fate of dietary3H-squalene in the rat

The absorption and metabolic fate of dietary squalene were investigated on the rat by administering a single oral dose of³H-squalene and¹⁴C-cholesterol. Experiments on rats with cannulated thoracic duct revealed that³H-squalene was, like¹⁴C-cholesterol, absorbed through the lymphatic vessels and that ca. 20% of absorbed³H-squalene was cyclized to sterols during the transit through the intestinal wall. Feces contained³H-sterols, indicating that newly synthesized mucosal sterols had been secreted into the gut lumen. In intact animals,³H-squalene appeared in the circulation more rapidly than¹⁴C-cholesterol and did not persist to any significant extent in the squalene-rich adipose and muscle tissues. The increase in dietary squalene load (8–48 mg) decreased the absorption percentage of³H-squalene (45–26%) but did not affect the absorption of¹⁴C-cholesterol (47%). Determination of fecal steroids revealed that during the first days absorbed³H-squalene was eliminated to a significantly higher extent than¹⁴C-cholesterol as fecal bile acids (34% vs 11%). The experiments indicate that the rat intestine has a marked capacity for absorbing dietary squalene and that the absorbed squalene is preferentially converted to bile acids in the liver.     

Fatty acid synthesis in rat testes injected intratesticularly or incubated with 1-14C acetate

Fatty acid synthesis was studied in testes of young and adult rats either injected intratesticularly or incubated with 1-¹⁴C acetate. The pattern of¹⁴C incorporation into lipids and individual fatty acids in the two age groups was similar but results obtained with intratesticular injection differed considerably from those obtained in the in vitro studies. In the former more than 70% of the¹⁴C incorporated in total lipids was in phosphatides, with about 15% in triglycerides and only minor amounts in cholesteryl esters and free fatty acids. Most of the¹⁴C incorporated into total fatty acids was in saturated acids (predominantly 16∶0). A small amount of¹⁴C was in the higher polyenes and there was a progressive increase with time after acetate injection in the¹⁴C content of 22∶5 W6. In testes incubated with 1-¹⁴C acetate, the phosphatide, triglyceride, and free fatty acid fractions had similar amounts of¹⁴C. In the total fatty acid fraction about 40% of the incorporated¹⁴C was in saturated acids (predominantly 14∶0 and 16∶0) and about 50% in the higher polyenes. Twenty carbon polyenes and 22∶5 W6 had significant¹⁴C incorporation, but most of the¹⁴C was in 22∶4 W6. About 80% of the¹⁴C in the latter compound was in the carboxyl carbon, indicating its origin from endogenous 20∶4 W6 elongated by the added 1-¹⁴C acetate used as substrate. The¹⁴C 22∶4 was present predominantly in the triglyceride and phosphatide fractions with minor amounts in other lipids.¹⁴C-labeled compounds of retention time greater than 22∶5 were also present in all lipid fractions. Presented at the ISF-AOCS World Congress Chicago, September 1970.     

<Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR characterization and stereochemical assignments of bile acids in aqueous media

The unconjugated bile acids cholic acid, deoxycholic acid, and chenodeoxycholic acid; their glycine and taurine conjugated glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and taurochenodeoxycholic acid; and a taurine conjugated ursodeoxycholic acid, tauroursodeoxycholic acid, were characterized through ¹H and ¹³C NMR in aqueous media under the physiological pH region (7.4±0.1). Assignments of ¹H and ¹³C signals of all the bile acids were made using a combination of several one- and two-dimensional, homonuclear (¹H−¹H) and heteronuclear (¹H−¹³C) correlations as well as spectral editing NMR methods. Stereochemical assignment of the five-membered ring of the bile acids is reported here for the first time. The complete characterization of various bile acids in aqueous media presented here may have implications in the study of the pathophysiology of biliary diseases through human biliary fluids using NMR spectroscopy.