Synthesis and storage

The liver is the principal site of synthesis of all circulating proteins apart from γ-globulins (produced in the reticuloendothelial system). The liver receives amino acids from the intestine and muscles and, by controlling the rate of gluconeogenesis and transamination, regulates plasma levels. Plasma contains 60–80 g/L of protein, mainly albumin, globulin and fibrinogen.

Albumin has a half-life of 16–24 days, and 10–12 g are synthesized daily. Its main functions are to maintain the intravascular oncotic (colloid osmotic) pressure, and to transport water-insoluble substances such as bilirubin, hormones, fatty acids and drugs. Reduced synthesis of albumin over prolonged periods produces hypoalbuminaemia and is seen in chronic liver disease and malnutrition. Hypoalbuminaemia is also found in hypercatabolic states (e.g. trauma with sepsis) and in diseases associated with an excessive loss (e.g. nephrotic syndrome, protein-losing enteropathy).

Transport or carrier proteins such as transferrin and caeruloplasmin, acute-phase and other proteins (e.g. α₁-antitrypsin and α-fetoprotein) are also produced in the liver.

The liver also synthesizes all factors involved in coagulation (apart from one-third of factor VIII) – that is, fibrinogen, prothrombin, factors V, VII, IX, X and XIII, proteins C and S and antithrombin (see Ch. 8) as well as components of the complement system. The liver stores large amounts of vitamins, particularly A, D and B₁₂, and lesser amounts of others (vitamin K and folate), and also minerals – iron in ferritin and haemosiderin and copper.

Degradation (nitrogen excretion)

Amino acids are degraded by transamination and oxidative deamination to produce ammonia, which is then converted to urea and excreted by the kidneys. This is a major pathway for the elimination of nitrogenous waste. Failure of this process occurs in severe liver disease.

Bile secretion and bile acid metabolism

Bile consists of water, electrolytes, bile acids, cholesterol, phospholipids and conjugated bilirubin. Two processes are involved in bile secretion across the canalicular membrane of the hepatocyte – bile salt-dependent and bile salt-independent processes – each contributing about 230 mL/day. Another 150 mL daily is produced by epithelial cells of the bile ductules.

Bile formation requires uptake of bile acids and other organic and inorganic ions across the basolateral (sinusoidal) membranes by multiple transport proteins (sodium taurocholate co-transporting polypeptide (NTCP) and sodium independent organic anion transporting polypeptide 2 (OATP2), Fig. 7.4). This process is driven by Na⁺/K⁺-ATPase in the basolateral membranes. Intracellular transport across hepatocytes is partly through microtubules and partly by cytosol transport proteins.

Figure 7.4 Cholesterol synthesis and its conversion to primary and secondary bile acids. All bile acids are normally conjugated with glycine or taurine. BSEP, bile salt export pump; MRP2, multidrug-resistant protein 2; MDR3, multidrug-resistant 3; OATP2, Na⁺ independent organic anion-transporting polypeptide 2; NTCP, Na⁺ taurocholate co-transporting polypeptide.

Bile acids are also synthesized in hepatocytes from cholesterol, the rate-limiting step being those catalysed mainly by cholesterol-7α-hydroxylase and the P450 enzymes (CYP7A1 and CYP8B1).

The bile acid receptor, farnesoid X, blocks bile acid formation from cholesterol and also regulates the transport proteins (NTCP, OATP2) that increase bile acid uptake by the liver. It is target for a new class of therapeutic drugs, farnesoid X receptor (FXR) agonists.

The canalicular membrane contains multispecific organic anion transporters, mainly ATPase dependent (ATP binding cassette), the multidrug-resistance protein 2 (MRP2), multidrug resistance protein (MDR3) and the bile salt excretory pump (BSEP), which carry a broad range of compounds including bilirubin diglucuronide, glucuronidated and sulphated bile acids and other organic anions against a concentration gradient into the biliary canaliculus. Na⁺ and water follow the passage of bile salts by diffusion across the tight junction between hepatocytes (a bile salt-dependent process). In the bile salt-independent process, water flow is due to other osmotically active solutes such as glutathione and bicarbonate.

Secretion of a bicarbonate-rich solution is stimulated mainly by secretin and is inhibited by somatostatin. This involves several membrane proteins, including the Cl⁻/HCO₃⁻ exchanger and the cystic fibrosis transmembrane conductance regulator which controls Cl⁻ secretion, and water channels (aquaporins) in cholangiocyte membranes.

The bile acids are excreted into bile and then pass via the common bile duct into the duodenum. The two primary bile acids – cholic acid and chenodeoxycholic acid (Fig. 7.4) – are conjugated with glycine or taurine, which increases their solubility. Intestinal bacteria convert these acids into secondary bile acids, deoxycholic and lithocholic acid. Figure 7.5 shows the enterohepatic circulation of bile acids.

Figure 7.5 Recirculation of bile acids. The bile salt pool is relatively small and the entire pool recycles six to eight times via the enterohepatic circulation. Synthesis of new bile acids compensates for faecal loss.

The average total bile flow is 600 mL/day. When fasting half flows into the duodenum and half is diverted into the gall bladder. The gall bladder mucosa absorbs 80–90% of the water and electrolytes, but is impermeable to bile acids and cholesterol. Following a meal, the I cells of the duodenal mucosa secrete cholecystokinin, which, stimulates contraction of the gall bladder and relaxation of the sphincter of Oddi, allowing bile to enter the duodenum. An adequate bile flow is dependent on bile salts being returned to the liver by the enterohepatic circulation.

Bile acids act as detergents; their main function is lipid solubilization. Bile acid molecules have both a hydrophilic and a hydrophobic end. In aqueous solutions they form micelles, with their hydrophobic (lipid-soluble) ends in the centre. Micelles are expanded by cholesterol and phospholipids (mainly lecithin), forming mixed micelles.

Bilirubin metabolism

Bilirubin is produced mainly from the breakdown of mature red cells by Kupffer cells in the liver and reticuloendothelial system; 15% of bilirubin is formed from catabolism of other haem-containing proteins, such as myoglobin, cytochromes and catalases.

Normally, 250–300 mg (425–510 mmol) of bilirubin are produced daily. The iron and globin are removed from haem and are reused. Biliverdin is formed from haem and then reduced to form bilirubin. The bilirubin produced is unconjugated and water-insoluble, due to internal hydrogen bonding, and is transported to the liver attached to albumin. Bilirubin dissociates from albumin and is taken up by hepatic cell membranes and transported to the endoplasmic reticulum by cytoplasmic proteins, where it is conjugated with glucuronic acid and excreted into bile. The microsomal enzyme, uridine diphosphoglucuronosyl transferase, catalyses the formation of bilirubin monoglucuronide and then diglucuronide. This conjugated bilirubin is water-soluble and is actively secreted into biliary canaliculi and excreted into the intestine within bile (Fig. 8.5). It is not absorbed from the small intestine because of its large molecular size. In the terminal ileum, bacterial enzymes hydrolyse the molecule, releasing free bilirubin which is then reduced to urobilinogen, some of which is excreted in the stools as stercobilinogen. The remainder is absorbed by the terminal ileum, passes to the liver via the enterohepatic circulation, and is re-excreted into bile. Urobilinogen bound to albumin enters the circulation and is excreted in urine via the kidneys. When hepatic excretion of conjugated bilirubin is impaired, a small amount is strongly bound to serum albumin and is not excreted by the kidneys; it accounts for the persistent hyperbilirubinaemia for a time after cholestasis has resolved.

Liver function tests

Liver biochemistry

Viral markers

Viruses are a major cause of liver disease. Virological studies have a key role in diagnosis; markers are available for most common viruses that cause hepatitis.

Haematological

A full blood count may show anaemia. The red cells are often macrocytic and can have abnormal shapes – target cells and spur cells – owing to membrane abnormalities. Vitamin B₁₂ levels are normal or high, while folate levels are often low owing to poor dietary intake. Other changes are caused by the following:

Biochemical

Immunological tests

Genetic analysis

These tests are performed routinely for haemochromatosis (HFE gene) and for α₁-antitrypsin deficiency. Markers are also available for the most frequent abnormal genes in Wilson’s disease (see p. 341).

Urine tests

Dipstick tests are available for bilirubin and urobilinogen. Bilirubinuria is due to the presence of conjugated (soluble) bilirubin, is found in patients with jaundice due to hepatobiliary disease, but is absent if unconjugated bilirubin is the major cause of jaundice. The presence of urobilinogen in urine in practice is of little value but suggests haemolysis or hepatic dysfunction.

Ultrasound examination

This is a non-invasive, safe and relatively cheap technique. It involves the analysis of the reflected ultrasound beam detected by a probe moved across the abdomen. The normal liver appears as a relatively homogeneous structure. The gall bladder, common bile duct, pancreas, portal vein and other structures in the abdomen can be visualized. Abdominal ultrasound is useful in:

Figure 7.6 Gall bladder ultrasound with multiple echogenic gallstones causing well-defined acoustic shadowing. (1) gall bladder; (2) gallstones; (3) echogenic shadow.

Other abdominal masses can be delineated and biopsies obtained under ultrasonic guidance.

Colour Doppler ultrasound will demonstrate vascularity within a lesion and the direction of portal and hepatic vein blood flow (Fig. 7.7).

Figure 7.7 Doppler signal of the portal vein is shown as a trace and the visual counterpart in red showing patency and forward flow into the liver.

Ultrasound contrast agents, mostly based on production of microbubbles within flowing blood, enhance the detection of vascularity, allowing the detection of abnormal circulation within liver nodules, giving a more specific diagnosis of hepatocellular carcinoma.

Hepatic stiffness (transient elastography). Using an ultrasound transducer, a vibration of low frequency and amplitude is passed through the liver, the velocity of which correlates with hepatic stiffness. Stiffness (measured in kPa) increases with worsening liver fibrosis (sensitivity and specificity 80–95% compared to liver biopsy). It is not accurate enough to diagnose cirrhosis, and less accurate for less severe fibrosis. It cannot be used in the presence of ascites and morbid obesity, and it is affected by inflammatory tissue and congestion. Acoustic radiation force impulse is incorporated into standard B mode ultrasonography and has similar physical principles to transient elastography.

Computed tomography (CT) examination

CT during or immediately after i.v. contrast shows both arterial and portal venous phases of enhancement, enabling more precise characterization of a lesion and its vascular supply (Fig. 7.8). Retrospective analysis of data allows multiple overlapping slices to be obtained with no increase in the radiation dose, providing excellent visualization of the size, shape and density of the liver, pancreas, spleen, lymph nodes and lesions in the porta hepatis. Multi-planar and three-dimensional reconstruction in the arterial phase can create a CT angiogram, often making formal invasive angiography unnecessary. CT also provides guidance for biopsy. It has advantages over US in detecting calcification and is useful in obese subjects, although US is usually the imaging modality used first to investigate liver disease.

Figure 7.8 Use of contrast-enhanced spiral CT. There is an irregular mass (arrow) in the posterior aspect of the right lobe of the liver which is only well seen on the early arterial phase enhanced scan.

Magnetic resonance imaging (MRI)

MRI produces cross-sectional images in any plane within the body and does not involve radiation. MRI is the most sensitive investigation of focal liver disease. Diffuse liver disease alters the T1 and T2 characteristics. Other fat-suppression modes such as STIR allow good differentiation between haemangiomas and other lesions. Contrast agents such as intravenous gadolinium, which allow further characterization of lesions, are suitable for those with iodine allergy, and provide angiography and venography of the splanchnic circulation. This has superseded direct arteriography.

Magnetic resonance cholangiopancreatography (MRCP)

This technique involves the manipulation of data acquired by MRI. A heavily T2-weighted sequence enhances visualization of the ‘water-filled’ bile ducts and pancreatic ducts to produce high-quality images of ductal anatomy. This non-invasive technique is replacing diagnostic (but not therapeutic) ERCP (see below), and is usually the next test if a biliary abnormality is present on US examination.

Plain X-rays of the abdomen

These are rarely requested but may show:

Radionuclide imaging – scintiscanning

In a ^99mTc-IODIDA scan, technetium-labelled iododiethyl IDA is taken up by the hepatocytes and excreted rapidly into the biliary system. Its main uses are in the diagnosis of:

Endoscopy

Upper GI endoscopy is used for diagnosis and treatment of varices, detection of portal hypertensive gastropathy, and for associated lesions such as peptic ulcers. Colonoscopy may show portal hypertensive colopathy. Capsule endoscopy can identify small intestinal varices.

Endoscopic retrograde cholangiopancreatography (ERCP)

This technique outlines the biliary and pancreatic ducts. It involves the passage of an endoscope into the second part of the duodenum and cannulation of the ampulla. Contrast is injected into both systems and the patient is screened radiologically. Contrast medium with a low iodine content of 1.5 mg/mL is used for the common bile duct so that gallstones are not obscured; a higher iodine content of 2.8 mg/mL is used for the pancreatic duct. Diagnostic ERCP has been replaced by MRCP in nearly all clinical settings. Therapeutic ERCP involves the following:

A raised serum amylase is often seen following ERCP and pancreatitis is the most common complication. Cholangitis with or without septicaemia is also seen, and broad-spectrum antibiotics (e.g. 500 mg ciprofloxacin × 2) should be given prophylactically to all patients with suspected biliary obstruction, or a history of cholangitis.

Percutaneous transhepatic cholangiography (PTC)

Under a local anaesthetic, a fine flexible needle is passed into the liver. Contrast is injected slowly until a biliary radicle is identified and then further contrast is injected to outline the whole of the biliary tree. In patients with dilated ducts, the success rate is near 100%. PTC is performed if ERCP fails or is likely to be technically difficult.

In difficult cases ERCP and PTC are sometimes combined, PTC showing the biliary anatomy above the obstruction, with ERCP showing the more distal anatomy. If an obstruction in the bile ducts is seen, a bypass stent can usually be inserted with or without temporary external biliary drainage. Contraindications of PTC are as for liver biopsy (see below). The main complications are bleeding and cholangitis with septicaemia, and prophylactic antibiotics should be given as for ERCP.

Angiography

This is performed by selective catheterization of the coeliac axis and hepatic artery. It outlines the hepatic vasculature and the abnormal vasculature of hepatic tumours, but spiral CT and magnetic resonance scanning have replaced diagnostic angiography. The portal vein can be demonstrated with increased definition using subtraction techniques replacing splenoportography (by direct splenic puncture).

In digital vascular imaging (DVI), contrast given intravenously or intra-arterially can be detected in the portal system using computerized subtraction analysis.

Hepatic venous cannulation allows abnormal hepatic veins to be diagnosed in patients with Budd–Chiari syndrome and is also used to measure portal pressure indirectly. There is a 1:1 relationship of occluded (by balloon) hepatic venous pressure, with portal pressure in patients with alcoholic or viral-related cirrhosis. The height of portal pressure has prognostic value for survival in cirrhosis, and a difference of the occluded minus the free hepatic venous pressure (hepatic venous pressure gradient HVPG) of 20% or more from baseline values or <12 mmHg, has been associated with protection from rebleeding, and prevention of other complications of cirrhosis.

Retrograde CO₂ portography is used when there is doubt about portal vein patency and can be combined with transjugular biopsy and hepatic venous pressure measurement.