Cholesterol via Bile Acids

Elimination of cholesterol from the human body takes place primarily by the fecal route as bile acids and neutral sterols, viz. cholesterol, coprosta-nol, and coprostanone. About one-third of cholesterol is normally catabo-lized by way of bile acids (11). As will be shown later, the amount of the latter depends on the body size, so that the weight correlates with the fecal bile acids, the average daily output of 250 mg corresponding to about 4 mg/ kg. The factors regulating hepatic bile acid production under normal conditions are, however, unknown in many respects. 

It seems evident that (1) if bile acid elimination is inhibited or impaired as a primary phenomenon, e.g., in biliary obstruction and hypercholesterolemia, a decreased catabolism of cholesterol leads to hypercholesterolemia and reduced cholesterol synthesis (2) if bile acid elimination is primarily augmented, e.g., after an external bile fistula, ileal bypass, ileal resection, cholestyramine treatment, or perhaps a diet rich in fibrous material, conversion of cholesterol to bile acids is enhanced, leading almost always, despite stimulated cholesterol synthesis, to a fall in serum cholesterol (3) if endogenous cholesterol production is primarily increased, e.g., by obesity and excess of calories, bile acid synthesis and elimination are augmented, preventing together with increased neutral sterol elimination in some but not all cases the increase of serum cholesterol. This suggests that removal, not production, of cholesterol is the primary factor which determines serum cholesterol level. 

There is no direct evidence so far that the actual concentration of serum cholesterol would determine bile acid production and elimination in man. For instance, increase of serum cholesterol by dietary cholesterol is not associated with compensatory increase in bile acid production (63,71,86,87). This does not exclude the possibility that an increase of some lipoprotein subfraction would stimulate bile acid synthesis. Thus determinations of bile acid synthesis by the isotope dilution method have shown markedly high values in triglyceridemic subjects (69), though according to sterol balance data this association is mostly determined by the degree of obesity of these patients (11,63). It is also interesting to note that though the serum cholesterol level and bile acid production are not normally correlated with each other, bile acid synthesis and the serum cholesterol pool are closely correlated in normocholesterolemic nonobese and obese subjects and in hypercholesterolemic individuals (88). 

An isolated defect in bile acid production has been found so far only in familial hypercholesterolemia (62), though even in this entity cholesterol catabolism as a whole may be decreased. Essential hypercholesterolemics (11) and hypothyroid patients (11,89) also tend to have a low bile salt elimination, though the excretion of cholesterol as such appears to decrease, too, particularly in the latter condition. In the circumstances in which bile salt elimination is decreased as a result of decreased hepatic function, elimination of cholesterol as such is also reduced (11). Under these conditions, serum cholesterol apparently increases only when the amount of elimination is decreased more than the feedback mechanism(s) are able to suppress synthesis, i.e., when the production exceeds elimination. 

Augmented Bile Salt Elimination in Interrupted Enterohepatic Circulation 

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The maximal capacity of human subjects to catabolize cholesterol via bile acids is not known, because complete external biliary fistula, ileal bypass. 

In hypothyroidism, the fecal steroids tended to contain relatively more bile acids (38 %) than normally (31%), while in hyperthyroidism quite normal values were obtained. This indicates that the hypercholesterolemia found consistently in hypothyroidism is caused by a defect in the elimination of cholesterol itself, the catabolism of cholesterol via bile acids being less significantly affected. 

Impairment of bile acid absorption and consequent loss of these acids via excretion presumably causes an increase in hepatic conversion of cholesterol to bile acids. This conversion lowers serum cholesterol, particularly when serum contains high levels of cholesterol derived from dietary intake. However, when fed with a cholesterol-free diet, 10% pectin supplementation stimulated a 3-fold increase in cholesterol biosynthesis (77). Biosynthesis of phospholipids and triglycerides also increased significantly hence, it was suggested that these increases occurred in response to diminished fat absorption occasioned by pectin intake. This compensatory biosynthesis of cholesterol and lipids may account for pectin s inability (in most cases) to lower serum cholesterol levels in animals fed cholesterol-free diets. 

A major pathway by which LDL are catabolized in hepatocytes and other cells involves receptor-mediated endocytosis. Cholesteryl esters from the LDL core are hydrolyzed, yielding free cholesterol for the synthesis of cell membranes. Cells also obtain cholesterol by de novo synthesis via a pathway involving the formation of mevalonic acid by HMG-CoA reductase. Production of this enzyme and of LDL receptors is transcriptionally regulated by the content of cholesterol in the cell. Normally, about 70% of LDL is removed from plasma by hepatocytes. Even more cholesterol is delivered to the liver via remnants of VLDL and chylomicrons. Thus, the liver plays a major role in the cholesterol economy. Unlike other cells, hepatocytes are capable of eliminating cholesterol by secretion of cholesterol in bile and by conversion of cholesterol to bile acids. 

An opposite effect is at the basis of the up-regulation of LDL receptors in response to treatments with bile acid sequestrants, intestinal cholesterol absorption inhibitors, and HMG-CoA reductase inhibitors. The first class of drugs inhibits the intestinal reabsorption of bile acids, thus promoting increased conversion of cholesterol to bile acids in the liver. The increased demand for cholesterol results in activation of the SREBP system and upregulation of LDL receptor synthesis (as well as cholesterol synthesis via upregulation of HMG-CoA reductase). Similarly, inhibition of intestinal cholesterol absorption with ezetimibe results in a reduction in the hepatic cholesterol pool... 

Peroxisomes are small granules arranged in clusters around the smooth ER and glycogen stores. They contain about 50 enzymes, some of which are used in respiration, purine catabolism and alcohol metabolism. They are responsible for about 20% of the oxygen consumption in the liver via a respiratory pathway that produces heat rather than ATP as its product. They differ from lysosomes in that they are not formed from outgrowths of the Golgi apparatus but are self-replicating, rather like mitochondria. They also play an important role in the metabolism of fatty acids as well as cholesterol and bile acid synthesis. 

In summary, these studies demonstrated that in CTX the impaired synthesis of bile acids is due to a defect in the biosynthetic pathway involving the oxidation of the cholesterol side-chain. As a consequence of the inefficient side-chain oxidation, increased 23, 24 and 25-hydroxylation of bile acid precursors occurs with the consequent marked increase in bile alcohol glucuronides secretions in bile, urine, plasma and feces (free bile alcohols). These compounds were isolated, synthesized and fully characterized by various spectroscopic methods. In addition, their absolute stereochemistiy determined by Lanthanide-Induced Circular Dichroism (CD) and Sharpless Asymmetric Dihydroxylation studies. Further studies demonstrated that (CTX) patients transform cholesterol into bile acids predominantly via the 25-hydroxylation pathway. This pathway involves the 25-hydroxylation of 5P-cholestane-3a,7a, 12a-triol to give 5P-cholestane-5P-cholestane-3a,7a,12a,25- tetrol followed by stereospecific 24S-hydroxylation to yield 5P-cholestane-3a,7a,12a,24S,25-pentol which in turn was converted to cholic acid. 

Differences in incorporation of radioactivity from the C-1 and C-2 carbons of propionate into cholesterol and bile acids by biliary fistula rats have been demonstrated. It appears that incorporation of propionate via HCO3" is not the major pathway. Feeding experiments with sodium [ C]bicarbonate produced no labelled cholesterol or bile acids. 

A study of the reduction of [24- C]3-oxo-5j8-cholanic acid in bile fistula rats given [l- Hjjethanol showed that all metabolites had a 3a-hydroxy group and all radioactive products (lithocholate, 3a,6/8-dihydroxy-5 -cholanate, chenodeoxycho-late and y8-muricholate) contained about 13 atom% excess deuterium in the 3/9 position. Thus, the 3)8-hydroxy-5/9-steroid dehydrogenase isoenzyme of alcohol dehydrogenase [172] has no function in the reductive metabolism of bile acids. Cholic acid was not radioactive but contained deuterium at the 3)8, 5)8 and other positions, probably because of the transfer of deuterium from ethanol via NADH to NADPH, which it utilized in the biosynthesis of cholesterol and bile acids and in oxido reduction of the 3-hydroxyl group of the latter [173]. 

As noted above, MeC trimerizes and MeLC does not self-associate in CHCI3. Under these conditions, Foster et al. [202] used vapor pressure osmometry to show that solubilized cholesterol (which dimerizes in CHCI3 [203]) heteroassociated with MeC but not with MeLC. The result was a 1 1 mixed dimer complex of cholesterol and MeC with a molar free energy of formation which was 33% that for the trimerization of MeC in the same solvent [202]. The bonding is presumably via the 3-hydroxyl functions in both steroids this interaction may be of potential importance in the binding of cholesterol to bile acids and salts within membranes and mixed micelles. 

Cholesterol molecules once formed in the body or absorbed from the diet can be eliminated from the human organism primarily by the gastrointestinal route as such or after conversion to bile acids. The latter are synthesized exclusively in the liver via a series of reactions which are initiated by 7a-hydroxylation of cholesterol (1). Bile acids subsequently formed are called primary bile acids, in contrast to secondary bile acids, which are formed by intestinal microorganisms from the primary ones during the enterohepatic circulation. 

Bile acids have two major functions in man (a) they form a catabolic pathway of cholesterol metabolism, and (b) they play an essential role in intestinal absorption of fat, cholesterol, and fat-soluble vitamins. These functions may be so vital that a genetic mutant with absence of bile acids, if at all developed, is obviously incapable of life, and therefore this type of inborn error of metabolism is not yet known clinically. A slightly decreased bile acid production, i.e., reduced cholesterol catabolism, as a primary phenomenon can lead to hypercholesterolemia without fat malabsorption, as has been suggested to be the case in familial hypercholesterolemia. A relative defect in bile salt production may lead to gallstone formation. A more severe defect in bile acid synthesis and biliary excretion found secondarily in liver disease causes fat malabsorption. This may be associated with hypercholesterolemia according to whether the bile salt deficiency is due to decreased function of parenchymal cells, as in liver cirrhosis, or whether the biliary excretory function is predominantly disturbed, as in biliary cirrhosis or extrahepatic biliary occlusion. Finally, an augmented cholesterol production in obesity is partially balanced by increased cholesterol catabolism via bile acids, while interruption of the enterohepatic circulation by ileal dysfunction or cholestyramine leads to intestinal bile salt deficiency despite an up to twentyfold increase in bile salt synthesis, to fat malabsorption, and to a fall in serum cholesterol. 

As already discussed, obesity is associated via augmented cholesterol synthesis with an increased bile acid production. Since obesity as such is a result of excessive consumption of calories, it is logical to infer that overeating stimulates cholesterol synthesis and secondarily bile acid production. An increased number and quantity of daily meals may, however, change under these conditions the metabolism of both cholesterol and bile acids in complicated ways which are not yet completely understood, by augmenting the number of enterohepatic circulations of bile salts. Increased intestinal contents and fecal mass may also interfere with reabsorption of bile acids. 

The major pathway of cholesterol elimination in the rat is via fecal excretion of bile acids. This pathway may be divided into two related events (a) the catabolism of cholesterol to bile acids and (b) the excretion of bile acids from the pool. Pituitary hormones may thus affect one or both of these processes. 

Excretion. Removal from the body occurs primarily via the conversion of cholesterol to bile acids. About 0.8 g of cholesterol is degraded daily by this method. Also, a minor amount is converted to the above mentioned hormones. Additionally, some cholesterol is never digested, and hence, excreted via the feces, particularly when the intake is high. 

Fig. 1 illustrates the excretion routes for cholesterol. The liver synthesizes cholesterol and secretes it into the biliary tract as cholesterol or bile acids. This amounts to somewhat less than 1 g/day. Minor losses occur in the urine as steroid hormones and via the skin as sebum and desquanmated epithelial cells. An important question addressed in Fig. 2 is how does the cholesterol absorbed from the diet affect these synthetic and excretory pathways There are 4 possibilities. First, as a patient changes from a cholesterol-free to a cholesterol-containing diet his hepatic cholesterol synthesis may decrease to compensate for the added exogenous load. Or the liver may simply re-excrete all of this dietary load plus the endogenously synthesized cholesterol and bile acids. Unfortunately, there are 2 remaining possibilities, namely. 

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