Las causales del procedimiento de ingreso como recaudación

Although RBC were ideal for studying the effects of phytosterols on membranes they could not be used to study specific effects of phytosterols on hepatocytes which might contribute to cholestasis. It was decided, therefore, to employ a tissue culture system using the human hepatocyte carcinoma cell line. Hep G2. The Hep G2 cell line was established by Knowles et al. (1980) from liver tumour biopsies and has been shown to demonstrate many liver specific functions including the synthesis and secretion of bile acids. Prior to describing the properties of Hep G2 and the effects of phytosterols which were observed, it is necessary to briefly review bile acid synthesis and the uptake and secretion of lipids by hepatocytes.

3.6.1 Bile Acid Synthesis

In healthy individuals cholesterol is converted in the liver to the two primary bile acids, cholic and chenodeoxycholic acid. This involves significant transformations to both the nucleus (hydroxylation and saturation of the C4-C5 double bond) and the side chain (oxidation) of the cholesterol molecule. A simplified overview of the classical pathway of bile acid synthesis is shown in Figure 3-6 (reviewed by Clayton, 1991).

The pathway begins with the conversion of cholesterol to 7a-hydroxycholesterol which is the rate limiting step. Synthesis proceeds via the metabolites, 5P-cholestane-3a,7a- diol and 5p-cholestane-3a,7a,12a-triol which possess a bile acid nucleus and a

cholesterol side chain. Oxidation of the side chain then starts with the conversion of the terminal methyl group to a carboxyl group forming the 27-carbon bile acids 3 a,7a- dihydroxy-5 P-cholestan-26-oic acid (DHCA) and 3a,7a, 12a-trihydroxy-5 P-cholestan- 26-oic acid (THCA). THCA and DHCA then undergo p-oxidation in the peroxisomes to produce chenodeoxycholic and cholic acids respectively. The bile acids are finally conjugated at the carboxylic carbon with the amino acids taurine or glycine. It should be noted that alternative pathways for bile acid synthesis exist. A discussion of these alternative pathways lies outside the scope of this thesis. Following conjugation, the bile acids are secreted into bile, where they participate in lipid digestion.

Cholesterol HO 7a-hydroxycholesterol HO OH 5p-cholestane- 3a,7a-diol OH 5p-cholestane- Qy 3a,7a,12a-triol HO OH _HO COOH _{OH COOH} DHCA THCA HO OH _{HO OH} Peroxisomal P-oxidation OH Chenodeoxycholic acid COOH COOH Cholic acid HO HO OH OH

3.6.2 Secretion of Biliary Lipids

Bile secretion is a unique and vital fiinction of the liver but very little is known about the mechanism at the cellular level. Bile consists of a complex mixture of water, inorganic ions, bile acids, cholesterol and phospholipid. This discussion, however, will be limited to the secretion of biliary lipids (Figure 3-7).

Basic Structure o f the Liver

Hepatocytes are arranged as plates one or two cells thick and radiate like the spokes of a wheel between the portal tracts and the central vein of the lobule o f the liver. These plates are separated by spaces, known as the sinusoids, which convey a mixture of hepatic arterial blood and portal venous blood from the portal tracts to the central vein. Between the hepatocytes are the bile canaliculi which transport the bile synthesised by the liver cells to the bile duct which then takes the formed bile to the gall bladder for temporary storage.

Bile Acids

Bile acids are the major driving force behind the secretion of bile. As already mentioned bile acids are synthesised from cholesterol. In humans, however, only 1-10% of the bile acids secreted into bile represents newly synthesised bile acids (Strange etal., 1984); most bile acids are derived from the enterohepatic circulation. The hepatic translocation o f bile acids from the blood to bile involves three distinct steps: i) hepatic uptake at the sinusoidal membrane of the hepatocyte, ii) transcellular processing and iii) excretion across the canalicular membrane.

Conjugated bile acids are taken up by the hepatocyte by a carrier mediated sodium- dependent co-transport process in which a sodium-taurocholate co-transporter drives the uptake o f bile acids. The energy input for this transporter is generated from the activity of a Na-K ATPase located in the sinusoidal membrane (Nathanson and Boyer,

1991). Unconjugated bile acids, however, appear to enter the liver cell independently of sodium coupling. These bile acids are thought to be transported by several different mechanisms some of which include dicarboxylic acid-anion exchange and facilitated diffusion (Nathanson and Boyer, 1991). Once within the hepatocyte the bile acids bind

to cytosolic binding proteins e.g. 3a-hydroxysteroid dehydrogenase (3a-HSD) and are rapidly translocated to the canalicular membrane where they are transported into bile by ATP-dependent transport systems (Muller et al., 1991).

Phospholipid

Biliary phospholipid is principally synthesised in the endoplasmic recticulum by

remodelling preformed lipid and consists almost totally of PC which possesses a unique fatty acid pattern with 16:0 at the 5«-l position and 18:1 or 18:2 at the sn-2 position (Hay et al., 1993). The PC molecules are transported from the endoplasmic recticulum through the cytoplasm and to the canalicular membrane via vesicles or by means of a phosphatidylcholine-transfer protein (PC-TP) (Cohen et al., 1994). From here it is believed that the PC are flipped from the inner to the outer leaflet of the canalicular membrane by the ATP dependent multidrug-resistance (MDR)-3 phospholipid translocase, creating a local excess of PC in the outer leaflet of the membrane. Bile acids which have been secreted into the canaliculi and which are unable to re-enter the hepatocyte then accumulate within the outer leaflet and promote vésiculation of the PC from the external leaflet of the canalicular membrane (Crawford et al., 1997).

Cholesterol

Almost all biliary cholesterol is non-esterified (Coleman, 1987). The majority of this cholesterol is not synthesised by the hepatocyte but originates from circulating plasma lipoproteins, principally low density lipoproteins (LDL). (For an overview of

lipoprotein uptake and metabolism see section 3 .10.1). Most of the free cholesterol released following lysosomal degradation of cholesterol esters distributes rapidly and widely throughout the liver cell but primarily to the plasma membrane. A small amount (approximately 1% each hour) of the remainder, however, is secreted into bile (Bilhartz and Dietschy, 1988). The mechanism for cholesterol secretion is unclear but is believed to involve trafficking via cytosolic binding proteins such as sterol carrier protein-2 (SCP-2) although other intracellular binding proteins may play a role (Puglielli et al.,

1995). On arrival at the canalicular pole of the hepatocyte it is thought that the

cholesterol simply diffuses through the membrane by a passive process completing the formation of the biliary micelles.

Plasma

Hepatocyte

Bile