CLINICAL LABORATORY DIAGNOSTICS OF LIVER PATHOLOGY

portal vein hepatic artery bile duct sinusoids bile canaliculi central vein LIVER STRUCTURE

LIVER FUNCTIONS MetabolicMetabolic -Carbohydrate metabolism -Lipid metabolism -Protein metabolism -Vitamin metabolism -Mineral metabolism ExcretoryExcretory -Biosynthesis of urea -Synthesis of bile DetoxicationDetoxication

Metabolism of carbohydrates in liver 1. Conversion of glucose-6- phosphate 2. Synthesis and decomposition of glycogen 3. Gluconeogenesis 4. Conversion of mannose, fructose and galactose to glucose

Lipid metabolism in liver Synthesis and decomposition of triacylglycerolsSynthesis and decomposition of triacylglycerols Synthesis and oxidation of fatty acidsSynthesis and oxidation of fatty acids Synthesis and decomposition of phospholipidsSynthesis and decomposition of phospholipids Synthesis and transformation of cholesterolSynthesis and transformation of cholesterol KetogenesisKetogenesis

Protein metabolism in liver Deamination, transamination and decarboxilation of amino acidsDeamination, transamination and decarboxilation of amino acids Synthesis of albumins of blood plasmaSynthesis of albumins of blood plasma Synthesis of majority of blood plasma globulinsSynthesis of majority of blood plasma globulins Synthesis of blood clotting factorsSynthesis of blood clotting factors

Vitamin metabolism in liver Formation of active form of vitamin DFormation of active form of vitamin D Formation of vitamin A from carotinsFormation of vitamin A from carotins Depo of cyanocobalamine and folic acidDepo of cyanocobalamine and folic acid Depo of vitamin EDepo of vitamin E Phosphorilation of vitamins B, formation of coenzyme formsPhosphorilation of vitamins B, formation of coenzyme forms

Detoxification in liver Substances detoxification in liver: -Xenobiotics -Products of protein decay in the intestine -The terminal products of metabolism

NORMAL METABOLISM OF BILE PIGMENTS CELLS OF RES Indirect bilirubin NADP + NADPH 2 Biliverdin reductase Biliverdin Iron Globin Verdoglobin NADP + Hemoxi- genase NADPH 2 Hemoglobin ERYTHROCYTES KIDNEYSKIDNEYS Stercobilinogen URINE Stercobilin Indirect bilirubin 1,7- 20,5 mkmol/l albumin Indirect bilirubin UDP-glucoronil- transferase Direct bilirubin mkmol/l BLOODBLOOD LIVERLIVER Bilirubin mono- glucoronid, 20 % Bilirubin di-glucoronid, 80 % Dipyrols  -glucoro- nidase Glucoronic acid Direct bilirubin BILEBILE INTESTINEINTESTINE Mesobilirubin Mesobilirubin (urobilinogen) Stercobilinogen Stercobilin STOOL

The decomposition of hemoglobin in tissues, bile pigments formation. After a life span of about 120 days the erythrocytes die. The dead erythrocytes are taken up by the phagocytes of the reticuloendothelial system of the body. About 7 gram of Hb is released daily from these phagocytosed erythrocytes. The Hb molecule is broken down into 3 parts:

(i) The protein (globin) part is utilized partly as such or along with other body proteins. (ii) The iron is stored in the reticuloendothelial cells and is reused for the synthesis of Hb and other iron containing substances of the body. (iii) The porphyrin part is converted to bile pigment, i.e. bilirubin which is excreted in bile.

Heme in the presence of the enzyme, heme oxygenase, loses one molecule of CO and one atom of iron in Fe 3+ form producing biliverdin. Biliverdin which is green in color is the first bile pigment to be produced; it is reduced to the yellow-colored bilirubin, the main bile pigment, by the enzyme biliverdin reductase

Bilirubin is a very toxic compound. For example, it is known to inhibit RNA and protein synthesis and carbohydrate metabolism in brain. Bilirubin formed in reticuloendothelial cells then is associated with plasma protein albumin to protect cells from the toxic effects. As this bilirubin is in complex with plasma proteins, therefore it cannot pass into the glomerular filtrate in the kidney; thus it does not appear in urine, even when its level in the blood plasma is very high. However, being lipid soluble, it readily gets deposited in lipid-rich tissues specially the brain.

This bilirubin is called indirect bilirubin or free bilirubin or unconjugated bilirubin.

The detoxication of indirect bilirubin takes place in the membranes of endoplasmatic reticulum of hepatocytes. Here bilirubin interact with UDP-glucuronic acid and is converted to the water soluble form - bilirubin mono- and diglucoronids. Another name of bilirubin mono- and diglucoronids is conjugated bilirubin or direct bilirubin or bound bilirubin. This reaction is catalized by UDP- glucoroniltransferase.

Conjugated bilirubin is water soluble and is excreted by hepatocytes to the bile. Conjugated (bound) bilirubin undergoes degradation in the intestine through the action of intestinal microorganisms. Bilirubin is reduced and, mesobilirubin is formed. Then mesobilirubin is reduced again and mesobilinogen is formed. The reduction of mesobilinogen results in the formation of stercobilinogen (in a colon). Stercobilinogen is oxidized and the chief pigment (brown color) of feces stercobilin is formed.

A part of mesobilinogen is reabsorbed by the mucous of intestine and via the vessels of vena porta system enter liver. In hepatocytes mesobilinogen is splitted to pyrol compounds which are excreted from the organism with bile. If the liver has undergone degeneration mesobilinogen enter the blood and is excreted by the kidneys. This mesobilinogen in urine is called urobilin, or true urobilin. Thus, true urobilin can be detected in urine only in liver parenchyma disease.

Another bile pigment that can be reabsorbed in intestine is stercobolinogen. Stercobolinogen is partially reabsorbed in the lower part of colon into the haemorroidal veins. From the blood stercobolinogen pass via the kidneys into the urine where it is oxidized to stercobilin. Another name of urine stercobilin is false urobilin.

The total bilirubin content in the blood serum is 1,7-20,5 micromol/l, indirect (unconjugated) bilirubin content is 1,7-17,1 micromol/l and direct (conjugated) bilirubin content is 0,86-4,3 micromol/l.

Differentiation between unconjugated and conjugated bilirubin. Direct and indirect bilirubin. Diazoreagent which is a mixture of sulfanilic acid, HCI and NaN0 2 is added to the serum. The conjugated bilirubin gives a reddish violet color with it and the maximum color intensity is obtained within 30 seconds; this is called direct test.

The unconjugated bilirubin does not give the direct test; however, it gives indirect test in which alcohol or caffeine is also added which sets free the bilirubin frum its complex with plasma proteins. Due to this difference in the type of diazo reaction given by these two forms of bilirubin, the term direct and indirect forms of bilirubin are also used

Jaundice or icterus is the orange-yellow discoloration of body tissues which is best seen in the skin and conjunctivae; it is caused by the presence of an excess of bilirubin in the blood plasma and tissue fluids. Depending upon the cause of an increased plasma bilirubin level, jaundice can be classified as (i) pre-hepatic, (ii) hepatic and (iii) post-hepatic

Pre-hepafic jaundice This type of jaundice is due to a raised plasma level of unconjugated bilirubin. It is due to an excessive breakdown of red cells which leads to an increased production of uncongugated bilirubin; it is also called haemolytic jaundice. As the liver is not able to excrete into the bile all the bilirubin reaching it, the plasma bilirubin level rises and jaundice results.

Hemolytic jaundice is characterized by Increase mainly of unconjugated bilirubin in the blood serum. Increased excretion of urobilinogen with urine. Dark brown colour of feces due to high content of stercobilinogen.

METABOLISM OF BILE PIGMENTS IN HEMOLYTIC JAUNDICE CELLS OF RES Indirect bilirubin albumin Indirect bilirubin Biliverdin reductase UDP-glucoronil- transferase Direct bilirubin NADP + NADPH 2 Biliverdin Iron Globin Verdoglobin NADP + NADPH 2 Hemoglobin Hemoxi- genase BLOODBLOOD LIVERLIVER Bilirubin mono- glucoronid, 20 % Bilirubin diglucoronid, 80 %  -glucoro- nidase Glucoronic acid Direct bilirubin BILEBILE ERYTHROCYTES KIDNEYSKIDNEYS INTESTINEINTESTINE STOOL Stool hypercholic URINE Urine dark Mesobilirubin Mesobilinogen (urobilinogen) Stercobilinogen Stercobilin Stercobilinogen Stercobilin Urobilin

Hepatic jaundice.This is typically seen in viral hepatitis. Several viruses are responsible for viral hepatitis and include hepatitis A, B, C and D viruses. The liver cells are damaged: inflammation produces obstruction of bile canaliculi due to swelling around them. This cholestasis causes the bile to regurgitate into the blood through bile canaliculi. The blood contains abnormally raised amount both of conjugated and unconjugated bilirubin and bile salts which are excreted in the urine.

Hepatic jaundice is characterized by 1.Increased levels of conjugated and unconjugated bilirubin in serum. 2.Dark coloured urine due to the excessive excretion of bilirubin and urobilinogen. 3.Pale, clay coloured stools due to the absence of stercobilinogen. 4.Increased activities of alanine and aspartate transaminases.

METABOLISM OF BILE PIGMENTS IN HEPATIC JAUNDICE CELLS OF RES Indirect bilirubin albumin Indirect bilirubin Biliverdin reductase UDP-glucoronil- transferase Direct bilirubin NADP + NADPH 2 Biliverdin Iron Globin Verdoglobin NADP + NADPH 2 Hemoglobin Hemoxi- genase BLOODBLOOD LIVERLIVER Bilirubin mono- glucoronid, 20 % Bilirubin diglucoronid, 80 %  -glucoro- nidase Glucoronic acid Direct bilirubin BILEBILE ERYTHROCYTES KIDNEYSKIDNEYS Urobilinogen INTESTINEINTESTINE STOOL Stool hypocholic URINE Urine dark Stercobi- linogen Stercobilin Bilirubin Urobilin Mesobilirubin Mesobilinogen (urobilinogen) Stercobilinogen Stercobilin

Post hepatic jaundice. This results when there is extrahepatic cholestasis due to an obstruction in the biliary passages outside the liver. In this way, the bile cannot reach the small intestine and therefore the biliary passages outside as well as inside the liver are distended with bile. This leads to damage to the liver and bile regurgitates into the blood.

Liver function tests will vary according to the degree of obstruction, i.e complete or incomplete. If the obstruction is complete, the stools become pale or clay-colored and the urine does not have any stercobilin. The absorption of fat and fat soluble vitamins also suffers due to a lack of bile salts. Excess of bile salts in the plasma produces severe pruritus (itching).

Obstructive (post hepatic ) jaundice is characterized by 1.Increased concentration mainly of conjugated bilirubin in serum. 2.Dark coloured urine due to elevated excretion of bilirubin and clay coloured feces due to absence of stercobilinogen.

METABOLISM OF BILE PIGMENTS IN OBSTRUCTIVE JAUNDICE CELLS OF RES Indirect bilirubin albumin Indirect bilirubin Biliverdin reductase UDP-glucoronil- transferase Direct bilirubin NADP + NADPH 2 Biliverdin Iron Globin Verdoglobin NADP + NADPH 2 Hemoglobin Hemoxi- genase BLOODBLOOD LIVERLIVER Bilirubin mono- glucoronid, 20 % Bilirubin diglucoronid, 80 % Bile acids  -glucoro- nidase Glucoronic acid Direct bilirubin BILEBILE ERYTHROCYTES KIDNEYSKIDNEYS Direct bilirubin INTESTINEINTESTINE STOOL Stool acholic, steatorhea URINE Direct bilirubin Bile acids Urine dark, foaming

FATTY LIVER

Spider naevus in liver cirrhosis in the ventral side of the left shoulder

Palmar erythema

Mild jaundice

Jaundiced patient