Liver Function

Liver Anatomy
- Liver is the largest organ in human body (~ 1.5 kg in adult). - It is located in the right upper quadrant of the abdomen and is attached by ligaments to the diaphragm. - It consists of two main lobes which are reddish brown in color.

Liver has a tremendous blood supply that reaches to the liver through 2 ways: a- portal vein which is formed from the superior mesenteric and splenic veins and so carries blood rich in nutrients from GIT. b- hepatic artery which carries oxygenated blood to the liver. Within the connective tissue of the liver, these vessels give off numerous small branches that form a net work around the so-called lobules.

The lobule is a hexagonal structure, measures 1-2 mm in diameter and forms the structural unit of the liver: a- It is formed from rows of hepatocytes radiate from the central hepatic vein and are separated by sinusoidal spaces, along the walls of which are interspersed hepatic macrophages, the Kupffer cells. b- Kupffer cells are part of the reticuloendothelial system and are phagocytic and so have an important detoxifying function. c- At the corners of each lobule are the portal tracts that contain branches of the hepatic artery, the portal vein and bile ducts. d- Blood flows from the portal tracts towards the central hepatic vein. Therefore: - hypoxia and toxins that are metabolized in the liver cause damage to the centrilobular area first; - toxins that do not depend on hepatic metabolism primarily affect the periphery of the lobule. e- The sinusoidal spaces: - are vascular channels that carry blood and they are located between cords of liver cells. - They are lined by endothelial cells and Kupffer cells. - These spaces received blood from the small branches of the hepatic artery and portal vein, located in the portal tract and blood from sinusoids and drains into the central hepatic veins and then to the central hepatic vein. Almost all nutrients from the gastrointestinal tract except fat micelles pass through the sinusoidal spaces before entering the systemic circulation. Distortion of this architectural arrangement, as in cirrhosis (fibrosis) of the liver, may allow blood to pass directly from the portal to the central and then into the hepatic vein, so bypassing the hepatocytes and therefore significantly reducing the detoxifying capacity.

FUNCTIONS OF LIVER
1- Metabolic functions a- When the glucose concentration is high in the portal vein, it is converted to glycogen and the carbon skeletons of fatty acids which are transported to adipose tissue as very low density lipoprotein (VLDL). b- During fasting the systemic plasma glucose concentration is maintained by the breakdown of glycogen (glycogenolysis) or by the synthesis Of glucose from substrates such as glycerol, lactate and amino acids (gluconeogenesis). c- Fatty acids reaching the liver from fat stores may be metabolized in the tricarboxylic acid cycle, converted to ketones or incorporated into triglycerides. 2- Synthetic functions Hepatocytes synthesize: a- plasma proteins (albumin &globulins), except immunoglobulins and complement. b- most coagulation factors, including fibrinogen and factors II (prothrombin), V, VII, IX, X, XI, XTI and XIII. Of these prothrombin (II) and factors VII, IX and X cannot be synthesized without vitamin K. c- the lipoproteins, VLDL and HDL. d- primary bile acids. The liver has a very large functional reserve. Deficiencies in synthetic function can only be detected if liver disease is extensive; such abnormalities are more often due to non-hepatic factors. For example, before a fall in plasma albumin concentration is attributed to advanced liver disease extrahepatic causes must be excluded, such as the loss of proteinthrough the kidney, gut or skin, or across capillary membranes into the interstitial space as in even mild inflammation or infection. Prothrombin levels, assessed by measuring the prothrombin time, may be reduced because of impaired hepatic synthesis, whether due to failure to absorb vitamin K or to hepatocellular damage. If hepatocellular function is adequate, parenteral administration of vitamin K will reverse the abnormality.

3- Excretion and detoxification: Substances that are inactivated and excreted by the liver include: a- Amino acids, which are deaminated in theliver. Amino groups, and the ammonia produced by intestinal bacterial action and absorbed into the portal vein, are converted to urea. b- Cholesterol, which is excreted in the bile either unchanged or after conversion to bile acids. c- Steroid hormones, which are metabolized and inactivated by conjugation with glucuronate and sulphate and excreted in the urine in these water-soluble forms. d- Many drugs, which are metabolized and inactivated by enzymes of the endoplasmic reticulum system; some are excreted in the bile. e- Toxins. The reticuloendothelial Kupffer cells in the hepatic sinusoids are well placed to extract toxic substances which have been absorbed from the gastrointestinal tract. f- Bilirubin. - Efficient excretion of the end-products of metabolism and of bilirubin depends on: normally functioning liver cells; normal blood flow through the liver; patent biliary ducts. - Efficient detoxification depends on the Kupffer cell function.

Bilirubin Metabolism
1. Bilirubin is derived mainly from the heme moiety of the hemoglobin

2.

3. 4.

5.

molecule liberated when effete red cells are removed from the circulation by the reticuloendothelial system. The iron in heme is reutilized but the 'tetrapyrrole ring is degraded to bilirubin. Other sources of bilirubin include myoglobin and the cytochromes Unconjugated bilirubin is not water-soluble; it is transported in the blood stream bound to albumin. In the liver, it is taken up by hepatocytes in a process involving specific carrier proteins. Bilirubin is then transported to the smooth endoplasmic reticulum where it undergoes conjugation, principally with glucuronic acid to form a diglucuronide; this process is catalyzed by the enzyme bilirubin-uridyl diphosphate glucuronyl transferase. Conjugated bilirubin is water-soluble and is secreted into the biliary canaliculi, eventually reaching the small intestine via the ducts of the

biliary system. 6. Secretion into the biliary canaliculi is the rate-limiting step in bilirubin metabolism. 7. In the gut, bilirubin is converted by bacterial action into urobilinogen (stercobilinogen), a colorless compound. Some urobilinogen is absorbed front the gut into the portal blood; hepatic uptake of this is incomplete, and a small quantity reaches the systemic circulation and is excreted in the urine. Most of the urobilinogen in the gut is oxidized in the colon to a brown pigment, urobilin, which is excreted in the stool. 8. In the absence of liver disease, no bilirubin is excreted in the urine. The bilirubin present in the blood is mainly unconjugated. Since it is protein-bound, it is not filtered by the renal glomeruli. 9. All the conjugated bilirubin is secreted into the biliary system and does not reach the blood stream. 10. Normal serum total bilirubin is 0.2-0.6 and up to 1mg/dl, while direct bilirubin is up to 0.3mg/dl (multiply by 17.1 to obtain mol/l).
The metabolism and excretion of bilirubin is summarized in figure.

Retention of Bilirubin (Hyperbilirubinemia): Jaundice

in

plasma

Jaundice, the yellow discoloration of tissues due to bilirubin deposition, is a frequent feature of liver disease. Jaundice only becomes clinically apparent when the plasma total bilirubin concentration reaches about 35 mol/L (2 mg/dl), twice the upper reference limit. It occurs when bilirubin production exceeds the hepatic capacity to excrete it. This may be because:
1. An increased rate of bilirubin production exceeds normal excretory

capacity of the liver (prehepatic jaundice). damaged liver cells (hepatic jaundice).

2. The normal load of bilirubin cannot be conjugated and/or excreted by

3. The biliary flow is obstructed, so that conjugated bilirubin cannot be

excreted into the intestine and is regurgitated into the systemic circulation (posthepatic jaundice).

1] Unconjugated hyperbilirubinemia (increase indirect bilirubin in serum): It occurs if there is: a marked increase in the bilirubin load as a result of hemolysis, or of the break-down of large amounts of blood after hemorrhage into the gastrointestinal tract or, for example, under the skin due to extensive bruising. impaired binding of bilirubin to ligandin (a binding protein in liver involved in conjugation of bilirubin) or impaired conjugation with glucuronate in the liver. In some pathological conditions plasma unconjugated bilirubin levels may increase so much that they exceed the protein-binding capacity. The lipid-soluble, unbound bilirubin damages brain cells (kernicterus).This is most likely to occur in newborn, particularly premature, infants in whom the hepatic conjugating mechanisms are immature. In addition, the proportion of unbound, unconjugated bilirubin, and therefore the risk of cerebral damage, increases if: plasma albumin concentrations are low; unconjugated bilirubin is displaced from binding sites by: high levels of free fatty acids; drugs such as salicylates or sulphonamides. Unconjugated bilirubin is normally all protein-bound, is not water soluble and therefore cannot be excreted in the urine. Patients with unconjugated hyperbilirubinemia do not have bilirubinuria i.e no bilirubin in urine ('acholuric jaundice'). 2] Conjugated bilirubinemia (increase direct bilirubin in serum): It is one of the earliest manifestations of impaired hepatic excretion. In most cases of jaundice in adults both conjugated and unconjugated fractions of bilirubin are increased in plasma but conjugated bilirubin predominates. Conjugated bilirubin is water-soluble, is less strongly protein-bound than the unconjugated form, and therefore can be excreted in the urine. Bilirubinuria is always pathological. Dark urine may be an early sign of some forms of hepatobiliary disease.

The following table summarizes the laboratory tests that are used to distinguish between the different types of jaundice, prehepatic, hepatic and posthepatic: Type Prehepatic Hepatic Poshepatic Etiology
-Increased hemolysis of RBCs at a rate faster than its excretion by liver. - Infective erythropioesis -Hepatitis e.g viral or drug induced. - Drugs e.g rifampicin which interfere with bilirubin conjugation. - Intrahepatic obstruction as in cirrhosis. - Gilberts syndrome. - Poisons as Ccl4, Pb, As. - Biliary obstruction - Gallstone. - Stricture. - Carcinoma of pancreas or biliary tree.

Total bilirubin Unconjugated bilirubin Conjugated bilirubin Urinary bilirubin Urinary urobilinogen Colour of urine Fecal stercobilinogen Colour of stool

Increased Increased Normal Absent Increased Dark yellow Increased Dark yellow

Increased Increased Increased Present Decreased Cola colour Decreased Light brown

Increased Normal Increased Present Absent Cola colour Absent Clay colour

Jaundice in the Newborn

Red-cell destruction, together with immature hepatic uptake, conjugation (due to decreased activity of hepatic glucouronyl transferase) and secretion of bilirubin, may cause a high plasma level of unconjugated bilirubin in the newborn; so-called 'physiological jaundice' is common. As a result of hemolytic disease, the plasma concentration of unconjugated bilirubin may be as high as 30 mg/dl and exceed the plasma protein-binding capacity; free unconjugated bilirubin may be deposited in the brain, causing kernicterus, a potential very serious disorder of the central nervous system. Treatment: 1. Phototherapy activates hepatic excretion of unconjugated bilirubin which may be due to converting bilirubin to another geometric isomers that can be more easily excreted in urine. 2. Administration of Phenobarbital that induces bilirubinconjugating enzymes.

Some Inherited Hyperbilirubinaemias

There is a group of inherited disorders in which either unconjugated or conjugated hyperbilirubinemia is the only detectable abnormality. 1- Gilbert's syndrome is a relatively common, familial condition which may be present at any age, but usually after the second decade. Plasma unconjugated bilirubin concentrations are usually between 1.2 and 2.5 mg/dl and rarely exceed 5 mg/dl. They fluctuate and may rise during intercurrent illness or fasting. The condition is harmless but must be differentiated from haemolysis and hepatitis. It often becomes evident when plasma bilirubin concentrations fail to return to normal after an attack of hepatitis, or during any mild illness which, because of the jaundice, may be misdiagnosed as hepatitis. 2- Crigler-Najjar syndrome, due to deficiency of hepatic glucuronyl transferase, is more serious. It usually presents at birth. The plasma unconjugated bilirubin concentration may increase to concentrations that exceed the binding capacity of albumin and so cause kernicterus.

Biochemical Tests Used in Diagnosis of Different Types of Liver Injury

The changes in plasma bilirubin concentration in various types of liver disorders have been already outlined. I] CELL DAMAGE Several tests are often misleadingly called 'liver function tests'. However, plasma enzyme activities indicate liver cell membrane damage rather than function. Because these enzymes are also present in other tissues, changes in plasma activities may reflect damage to those tissues rather than to the liver. Transaminases (alanine (ALT GPT) and aspartate (AST; GOT) aminotransferases): A rise in plasma transaminase activities is a sensitive indicator of damage to cytoplasmic and/or mitochondrial membranes. Plasma enzyme activities rise when the membranes of only very few cells are damaged. Liver cells contain more AST than ALT, but ALT is confined to the cytoplasm in which its concentration is higher than that of AST which is present in cytoplasm and mitochondria. In inflammatory or infective conditions, such as viral hepatitis, the cytoplasmic membrane sustains the main damage; leakage of cytoplasmic contents causes a relatively greater increase in plasma ALT than AST activities. In infiltrative disorders in which there is damage to both mitochondrial and cytoplasmic membranes, there is a proportionally greater increase in plasma AST activity than ALT. The relative plasma activities of ALT and AST may help to indicate the type of cell damage.

Aspartate aminotransferase (AST)

AST (glutamate oxaloacetate transaminase, GOT) is present in high concentrations in cells of cardiac and skeletal muscle, liver, kidney and erythrocytes. Damage to any of these tissues may increase plasma AST levels. Causes of raised plasma AST activities: 1- Artefactual: due to in vitro release from erythrocytes if there is hemolysis or if separation of plasma from cells is delayed.

2- Physiological: during the neonatal period (about 1.5 times the upper

adult reference limit). 3- Marked increase (10 to 100 times the upper adult reference limit) occurs in circulatory failure with 'shock' and hypoxia; myocardial infarction; acute viral or toxic hepatitis. 4- Moderate increase: a-cirrhosis (may be normal, but may rise to twice the upper adult reference limit); b- cholestatic jaundice (up to 10 times the upper adult reference limit); c- malignant infiltration of the liver (may be normal, but may rise to twice the upper reference limit); d- after trauma or surgery (especially after cardiac surgery).

Alanine Transaminase (ALT)

ALT (glutamate pyruvate transaminase, GPT) is present in high concentrations in liver and, to a lesser extent, in skeletal muscle, kidney and heart. Causes of raised plasma ALT activities: 1. A marked increase (10 to 100 times the upper limit of the adult reference range): a- circulatory failure with 'shock' and b- hypoxia; acute viral or toxic hepatitis. 2- Moderate increase: a- cirrhosis (may be normal or up to twice the upper adult reference limit); b- cholestatic jaundice (up to 10 times the upper reference limit in adults); II- SYNTHETIC FUNCTION The measurement of the prothrombin time and plasma albumin concentration may be used to assess function in certain conditions. The hepatic synthetic and secretory capacities are large; only severe and usually prolonged liver disease demonstrably impairs prothrombin and albumin synthesis. Albumin is synthesized in the liver and its concentration in the serum is in part a reflection of the functional capacity of the organ. Hypoalbuminemia is such a common finding in many severe illnesses that it is a less specific indicator of impaired synthetic capacity than a prolonged prothrombin time.

The prothrombin time may be prolonged due to cholestasis; fat soluble vitamin K cannot be absorbed normally if fat absorption is impaired due to intestinal bile salt deficiency. The abnormality is then corrected by parenteral administration of the vitamin. A prolonged prothrombin time may also result from severe impairment of synthetic ability if the liver cell mass is greatly reduced; in such cases it is not corrected by parenteral administration of vitamin K.

III- EXCRETORY FUNCTION A high plasma conjugated bilirubin concentration indicates impaired hepatic excretory function due to biliary obstruction (cholestasis); this may be accompanied by a high plasma alkaline phosphatase (ALP) activity and gamma-glutamyl transferase (GGT).

Alkaline Phosphatase
The alkaline phosphatases are a group of enzymes that hydrolyse organic phosphates at high pH. a- They are present in most tissues but are in particularly high concentration in the osteoblasts of bone and the cells of the hepatobiliary tract, intestinal wall, renal tubules and placenta. b- The exact metabolic function of ALP is unknown but it is probably important for calcification of bone. c- In adults plasma ALP is derived mainly from bone and liver in approximately equal proportions; the proportion due to the bone fraction is increased when there is increased osteoblastic activity that may be physiological. Causes of raised plasma ALP activity: 1. Physiological: During the last trimester of pregnancy, in preterm infants and in growing children during the pubertal bone growth spurt. There is a gradual increase in the proportion of liver ALP with age. 2. Bone disease: rickets and osteomalacia. 3. Liver disease: intra- or extrahepatic cholestasis. Plasma activities rise in cholestatic liver disease because ALP synthesis is increased and the enzyme within the biliary tract is regurgitated into plasma. Isoenzymes of ALP

Bone disease with increased osteoblastic activity, or liver disease with involvement of the biliary tracts, are the commonest causes of an increased total alkaline phosphatase activity. Rarely the cause is not apparent and further tests may be helpful. The isoenzymes originating from cells of bone, liver, intestine and placenta may be separated by electrophoresis, but interpretation may be difficult if the total activity is only marginally raised. The placental isoenzyme is more stable at 65oC than the bone, liver and intestinal isoenzymes, and heat inactivation may help to differentiate the heat-stable from the heat-labile fraction.

Gamma glutamyl transferase (GGT)

Gamma glutamyl transferase occurs mainly in the cells of liver (derived from the endoplasmic reticulum of the cells of the hepatobiliary tract) in addition to kidneys, pancreas and prostate. Plasma GGT activity is higher in men than in women. Causes of raised plasma GGT activity 1. Cholestatic liver disease, when changes in GGT activity usually parallel those of alkaline phosphatase. 2. Hepatocellular damage, such as that due to infectious hepatitis; measurement of plasma transaminase activities is a more sensitive indicator of such conditions. 3. Induction of enzyme synthesis, without cell damage, by drugs or alcohol. Many drugs, most commonly the anticonvulsants, phenobarbitone and phenytoin, and alcohol induce proliferation of the endoplasmic reticulum.
o Slightly or moderately raised activities (up to about three times the

upper reference limit) are particularly difficult to interpret. o Very high plasma GGT activities, out of proportion to those of the transaminases, may be due to: alcoholic hepatitis; induction by anticonvulsant drugs or by chronic alcohol intake; cholestatic liver disease.

In conclusion Biochemical investigations can be used to investigate hepatic disorders, the mechanisms underlying which can be divided into three main groups; these often coexist, but one usually predominates in any particular condition. 1. Liver-cell damage is characterized by release of enzymes (aspartate (AST; GOT) and alanine (ALT; GPT) transaminases) from damaged hepatocytes. Plasma ALT and AST activities are increased. 2. Cholestasis is characterized by retention of conjugated bilirubin and of alkaline phosphatase (ALP), and by increased ALP synthesis at the sinusoidal surface. Plasma conjugated bilirubin levels and alkaline phosphatase activities are increased. 3. Reduced mass of functioning cells, if considerable, is characterized by a reduction in prothrombin and albumin synthesis. The prothrombin time is prolonged and plasma albumin concentration is reduced.

DISEASES OF THE LIVER I- Cholestasis

Cholestasis may be either: 1. intrahepatic, in which bile secretion from the hepatocytes into the canaliculi is impaired, due to: a- viral hepatitis; b- drugs such as chlorpromazine or toxins such as alcohol; c- inflammation of the biliary tract (cholangitis); 2. extrahepatic, due to obstruction to the flow of preformed bile through the biliary tract by: a- biliary stones; b- inflammation of the biliary tract; c- pressure on the tract from outside by malignant tissue, usually of the head of the pancreas. Unconjugated Bilirubin concentrations in plasma is usually increased but it may be normal if only part of the biliary system is involved by intrahepatic lesions. Bilirubin can be secreted by the unaffected areas. Alkaline phosphatase activity is the most sensitive test for cholestasis. Increased synthesis of ALP in the affected ducts increases the activity of this enzyme in plasma. Patients with prolonged, and more widespread cholestasis may present with severe jaundice and itching due to deposition of retained bile salts in the skin; the plasma bilirubin concentration may be as high as 50mg/dl.

II- Acute Hepatitis

The biochemical findings in acute hepatitis are predominantly those of cell membrane damage with an increase in plasma ALT activity greater than that of AST. Viral hepatitis is most commonly used to describe three principal types of viral infection in which the clinical features of the acute illness are very similar, although with a different incubation period. 1. Hepatitis A ('infectious hepatitis) transmitted by the faecal-oral route as a foodborne infection, is relatively common in schoolsand other institutions and has an incubation period of between 15 and 45 days. Relapses may occur but it rarely progresses to chronic hepatitis. 2. Hepatitis B ('serum hepatitis) is transmitted by blood products and other body fluids; it occurs more sporadically than hepatitis A. It has a longer incubation period of between 40 and 180 days. Some patients may be anicteric, some may develop fulminant hepatitis or chronic active hepatitis and later cirrhosis. They may become asymptomatic carriers of the disease. 3. Hepatitis C (non-A, non-B hepatitis), which is usually the result of transfusion of blood or blood products, has an incubation period of between 15 and 50 days. About half the patients develop chronic hepatitis sometimes progressing to cirrhosis. In all types there may be a three or four day history of anorexia, nausea and tenderness or discomfort over the liver before the onset of jaundice. Some patients remain anicteric. Plasma transaminase activities are very high from the onset of symptoms; they peak about four days later, when jaundice becomes detectable but may remain elevated for several months. Once jaundice appears some of the initial symptoms improve.

III Chronic Hepatitis

The finding of persistent usually only slightly, raised plasma transaminase activities, sometimes with chronic or recurrent symptoms suggesting liver disease, may be caused by several disorders. It may be the only abnormal biochemical finding. 1- Chronic persistent hepatitis It is a term used to describe the finding of slightly raised plasma transaminase activities without clinical signs or symptoms and without a significant change in their activities over many years. They rarely exceed three times the upper reference limits. Jaundice is unusual. The biochemical findings may be discovered by a chance. The condition is probably benign. 2- Chronic active hepatitis It is caused by active hepatocellular destruction with episodes of relapses and remissions. It may progress to cirrhosis. It occurs at any age but is most common in young women. It may be associated with, or a consequence of viral infections, such as HBV or HCV or may be drug-induced; As the disease progresses more cells are destroyed and the plasma AST activity may rise to or exceed that of ALT, slight jaundice may develop. If there is significant hepatocellular destruction the plasma albumin concentration falls.

IV Cirrhosis
Cirrhosis is the end result of many inflammatory and metabolic diseases involving the liver, including prolonged toxic damage most usually due to alcohol. Anything that damages liver cells can cause cirrhosis, such as chemical toxins, severe malnutrition, viruses, or metabolic disorders. Damaged or dead liver cells are replaced by fibrous scar tissue, leading to a breakdown in liver function and eventually resulting in liver failure and death.

 If the cause of the damage is removed early enough, the liver is capable of regenerating itself and will heal. If there is too much damage, a liver transplant may be required.

In the early stages there may be no abnormal biochemical findings. During phases of active cellular destruction the plasma AST, and sometimes ALT, activities rise slightly. In advanced cases, the biochemical findings are mostly associated with a reduced functioning cell mass.

V Hepatocellular Failure

Liver damage severe enough to cause obvious clinical signs of impaired hepatocellular function. It may be caused by severe hepatitis, advanced cirrhosis, or follow an overdose of a liver toxin such as paracetamol (acetaminophen). The biochemical findings may include any or all of those of acute hepatitis. Jaundice is progressive. In the final stage the number of hepatocytes, and so the total amount of transaminases released, may be so reduced that plasma activities fall despite continuing damage to the remaining cells. This finding should not be interpreted as a sign of recovery. Impaired hepatic deamination of amino acids, causing accumulation of amino acids in plasma with overflow aminoaciduria and sometimes hyperammonaemia.

Treatment of End Stage Liver Disease

Liver transplantation may be the only possible treatment for end stage liver disease. Complications include graft failure, hepatic artery thrombosis, infection and acute and chronic rejection. Both acute rejection, which occurs in up to 80 per cent of recipients, and chronic rejection, occurring in about 10 per cent of cases, are associated with a rise in plasma bilirubin concentration and alkaline phosphatase activity, chronic rejection is usually irreversible.

Bile and Gall Stone

1- Formation of bile Between one and two litres of bile are produced by the liver daily. This hepatic bile contains bilirubin, bile salts, phospholipids and cholesterol, as well as electrolytes in concentrations similar to those in plasma. Small amounts of protein are also present. In the gallbladder there is active reabsorption of sodium, chloride and bicarbonate, together with an isosmotic amount of water. Consequently, gallbladder bile is ten times more concentrated than hepatic bile; sodium is the major cation and bile salts the major anions. The concentrations of other nonabsorbable molecules, such as conjugated bilirubin, cholesterol and phospholipids, also increase. Gall Stones

Although most gallstones contain all biliary constituents, they consist predominantly of one. Only about 10 per cent contain enough calcium to be radiopaque and in this way differ from renal calculi. 1- Pigment stones are found in such chronic hemolytic states. a- Increased breakdown of hemoglobin increases bilirubin formation and therefore biliary secretion. b- The stones consist mostly of bile pigments, with variable amounts of calcium. c- They are small, hard and dark green or black, and are usually multiple. 2- Cholesterol stones. a- Cholesterol is most likely to precipitate if bile is supersaturated with it; further precipitation on a nucleus of crystals causes progressive enlargement. b- Not all patients with a high biliary cholesterol concentration suffer from bile stones. c- There is no association hetween hypercholesterolemia and the formation ofcholesterol gall stones. However, there is an increased incidence in patients taking some lipid lowering drugs, such as the fibric acid derivative, clofibrate. 3- Mixed stones. a- Most gall stones contain a mixture of bile constituents, usually with a cholesterol nucleus as a starting point. b- They are multiple, faceted, dark brown stones with a hard shell and a softer centre.