مرحلة ثالثة الدراسة الصباحية Clinical Biochemistry
Liver function tests
The liver is the largest organ in the body It is located below the diaphragm in the right upper quadrant of the abdominal cavity and extended approximately from the right 5th rib to the lower border of the rib cage. The liver is separated into a right and left lobe, separated by the falciform ligament. The right is much larger than the left.
Functions of liver ① Excretory function: bile pigments, bile salts and cholesterol are excreted in bile into intestine. ② Metabolic function: liver actively participates in carbohydrate, lipid, protein, mineral and vitamin metabolisms. ③ Hematological function: liver is also produces clotting factors like factor V, VII. Fibrinogen involved in blood coagulation is also synthesized in liver. It synthesizes plasma proteins and destruction of erythrocytes. ④ Storage functions: glycogen, vitamins A, D and B12, and trace element iron are stored in liver. ⑤ Protective functions and detoxification: Ammonia is detoxified to urea. kupffer cells of liver perform phagocytosis to eliminate foreign compounds. Liver is responsible for the metabolism of xenobiotic.
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Classification of liver functions test Classified based on the major functions of liver: ① Excretion: Measurement of bile pigments, bile salts. ② Serum enzymes: Transaminase (ALT, AST), alkaline phosphate (ALP), 5’-nucleotidase, LDH isoenzyme. ③ Synthetic function: Prothrombin time, serum albumin. ④ Detoxification :
Excretion: Bilirubin Bilirubin is the main bile pigment that is formed from the breakdown of heme in red blood cells. The broken down heme travels to the liver, where it is secreted into the bile by the liver. Serum bilirubin Normally, a small amount of bilirubin circulates in the blood. Serum bilirubin is considered a true test of liver function, as it reflects the liver's ability to take up, process, and secrete bilirubin into the bile. A. indirect bilirubin (Normal value = 0.3 - 1.2 mg/dl) B. direct bilirubin (Normal value ≤ 0.4 mg/dl) C. total bilirubin Normal value for = 0.3- 1.2 mg/dl.
A. urobilinogen : Conjugated bilirubin is excreted via bile salts to intestine. Bacteria in the intestine break down bilirubin to urobilinogen for excretion in the feces (normal value for fecal urobilinogen = 40 - 280 mg/day) Normally there are mere traces of urobilinogen in the urine. Average is 0.64 mg , maximum normal 4mg/24hours.
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B. Urobilin Urobilin is the final product of oxidation of urobilinogen by oxygen in air. The amount change with the amount of urobilinogen excretion.
B. bilirubinurine: Bilirubin is not normally present in urine and feces since bacteria in intestine reduce it to urobilinogen. The kidneys do not filter unconjugated bilirubin because of its avid binding to albumin. Conjugated bilirubin can pass through glomerular filter. Bilirubin is found in the urine in obstructive jaundice due to various causes and in cholestasis. Note: Bilirubin in the urine may be detected even before clinical jaundice is noted.
Who is a candidate for the test? Bilirubin is used to diagnosis of jaundice. Abnormal bilirubin levels can be found in many disorders, including: blocked bile ducts, cirrhosis, hepatitis and other liver diseases or immature liver development in newborns. Hemolytic Jaundice Hepatic Jaundice Obstructive jaundice ( Cholestasis) Congenital Jaundice
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Serum enzymes A large number of enzyme estimations are available which are used to ascertain liver function. They are being divided into two groups:
I: most commonly and routinely done in the laboratory.
Serum transaminase(ALT/AST) Serum alkaline phosphate (ALP)
II: not routinely done in the laboratory.
Alanine transaminase (ALT)
ALT or sGPT (serum glutamate pyruvate transaminase Aspartate aminotransferase (AST)
AST or sGOT (serum glutamate oxaloacetate transaminase) Therefore, when the liver is injured, GPT is released into the bloodstream.
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Elevated levels of GPT may indicate: alcoholic liver disease cancer of the liver cholestasis or congestion of the bile ducts cirrhosis or scarring of the liver with loss of function death of liver tissue Hepatitis or inflammation of the liver noncancerous tumor of the liver Use of medicines or drugs toxic to the liver GOT also reflects damage to the hepatic cells and is less specific for liver disease. It can also be released with heart, muscle and brain disorders. Therefore, this test may be ordered to help diagnose various heart, muscle or brain disorders, such as a myocardial infarct (heart attack).
Elevated levels of GOT may indicate : acute hemolytic anemia, Acute pancreatitis or inflammation of the pancreas. Acute renal failure or loss of kidney function. Cirrhosis of the liver. Hepatitis heart attack primary muscle disease recent surgery severe burns Muscle injury
Although GOT is not a specific for liver as the GPT, ratios between GPT and GOT are useful to physicians in assessing the etiology of liver enzyme abnormalities. ◆ Normally : GPT is normal, GOT is normal, GPT/GOT is about 1.15. ◆ Virus hepatitis: GPT↑, GOT is normal, GPT/GOT＞1, even more than 2.5； ◆ Chronic hepatitis: GPT↑, GOT ↑GPT/GOT is about 1.
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◆ Liver cancer, cirrhosis, Alcohol-induced hepatitis: GPT↑, GOT ↑ < 1, about 0.6~0.7. ◆ Accute myocardial infarct :< 1 GPT and GOT is in the different distribution of the hepatocytes. GPT exists primarily in the cytoplasm of liver cell. If there is a slight liver cell damage, GPT firstly leak into the bloodstream, so that the serum GPT increased. The GOT mainly in the "mitochondria“of liver cells, the mitochondria are "bubble" in the liver cell cytoplasm. If there is a slight liver cell damage, GOT don`t leak into the bloodstream.
Alkaline phosphatase (ALP)
ALP occurs in all tissues, especially liver and bone. The alkaline phosphatase test is often used to help diagnose certain liver diseases and bone disorders. Nor mal range: 30 - 95 IU/L (3-13 kings unit) ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. most effective in an alkaline environment In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. Levels are significantly higher in children and pregnant women.
Higher levels of ALP than normal may indicate:
liver disease bone disease leukemia, a cancer of the blood and bone marrow various hormone problems Pregnancy
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Lower levels of ALP than normal may indicate:
anemia, or a low red blood cell count malnutrition Various hormone problems
Determination of total plasma proteins/ albumin/ globulin/ A : G ratio This yields most useful information in chronic liver disease. Liver is the site of albumin synthesis and also possibly of some of α and β globulins. Normal value: Total plasma proteins: 80~110mg/dl Albumin: 40-50mg/dl Globulin: 25~35mg/dl A: G ratio: 1.5~2.5 Serum Albumin
Albumin is an important blood protein that is made only by the liver and excreted ! by the kidneys.
Albumin is essential for maintaining the osmotic pressure in the vascular system. ! Low albumin level produce ascites.
★ Albumin is also very important in the transportation of many substances such ! as drugs, lipids, hormones and toxins that are bound to albumin in the bloodstream. This test is normally performed to assist in diagnosing diseases that affect proteins ! in the body, such as cancer, liver disease, renal or intestinal problems, and immune disorders.
Normal range: 34 - 54 g/L ! Abnormally low contents of albumin may indicate: ascites extensive burns kidney disease liver disease malabsorption syndromes 7 م.م. وفاء حفظي عجام
Formation of prothrombin by liver At least 12 different proteins are involved in clotting. Blood clotting factors are proteins made by the liver and are associated with the incorporation of vitamin K metabolites into a protein. When the liver is significantly injured, these proteins are not normally produced. PT (Prothrombin time)
Estimation of plasma fibrinogen APTT(activated partial thromboplastin time)
Prothrombin time (protime or PT) Prothrombin is a plasma protein that is converted into thrombin during blood clotting. ★ Prothrombin is formed in the liver from inactive “preprothrombin” in presence of vitamin K. What is prothrombin time? prothrombin time is measured as prothrombin activity. The term prothrombin time was given to time required for clotting to take place in plasma to III factor and Ca+ have been added. PT is used to assess the activity of extrinsic blood clotting pathway. ★ PT is also a useful test of liver function, since there is a good correlation between abnormalities in coagulation measured by PT and the degree of liver dysfunction. PT is usually expressed in seconds and compared to a normal control patient’s blood.
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Formation of Bilirubin from Heme•
Heme is degraded in RE system (esp. liver & spleen) 85% from RBCs 15% from turnover of immature RBCs & cytochromes Hemoglobin (Hb) consists of four (tetramer) sub-units held together by multiple non- covalent interactions. Each subunit consists of heme (Protoporphyrin IX and ferrous ion (Fe+2) and globin protein. Globin protein folds around heme group forming hydrophobic pocket that protects the heme , which is the site of oxygen binding. RBC is largest repository of heme in humans which it is life span about 120 days.
Heme
↓ hemeoxygenase
biliverdin(green) ↓
bilirubin (red-orange) bile pigments ↓ In Blood with albumin UNCONJUGATED BILIRUBIN (Or INDIRECT BILITUBIN)
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Bilirubin Metabolism in the Liver•
Uptake of Bilirubin by hepatocytes:•
Bilirubin dissociates from its carrier albumin & enters hepatocytes
Conjugation of Bilirubin:•
In hepatocytes, bilirubin is conjugated with two molecules of glucuronic acid by the enzyme glucuronyltransferase
Excretion of bilirubin into bile:•
Conjugated bilirubin (bilirubin diglucuronide) is transported into bile canalculi& then into bile. Process is energy dependent & is impaired in liver diseases
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Bilirubin Metabolism in the Intestine•
Conjugated bilirubin ↓ bacteria in the intestine Urobilinogen
Stercobilin Reabsorbed in stool (brown) ↓ Kidney ↓ Urine Urobilin (yellow)
NORMAL BILIRUBIN METABOLISM
Uptake of bilirubin by the liver is mediated by a carrier protein (receptor)
Uptake may be competitively inhibited by other organic anions
On the smooth, bilirubin is conjugated with glucoronic acid, xylose, or ribose
Glucoronic acid is the major conjugate - catalyzed by UDP glucuronyltranferase
“Conjugated” bilirubin is water soluble and is secreted by the hepatocytes into the biliary canaliculi
Converted to stercobilinogen (urobilinogen) (colorless) by bacteria in the gut
Oxidized to stercobilin which is colored
Excreted in feces
Some stercobilin may be re-adsorbed by the gut and re-excreted by either the liver or kidney
HYPERBILIRUBINEMIA Increased plasma concentrations of bilirubin (> 3 mg/dL) occurs when
There is an imbalance between its production and excretion Recognized clinically as jaundice
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Physiologic Hyperbilirubinemia •
Increased production •
Short RBC lifespan –
Increased shunt bilirubin –
Decrease clearance •
Portal vein shunting via ductusvenosus –
Decreased conjugation •
Decreased UDPGA synthesis –
Decreased UDPG transferase –
Increased enterohepatic circulation •
High concentration of bilirubin in meconium –
Decreased bowel motility – Causes of Hyperbilirubinemia and Jaundice
CAUSES of UNCONJUGATED (INDIRECT) HYPERBILIRUBINEMIA
1. Increased lysis of RBCs (i.e., increased hemoglobin release) •Isoimmunization (blood group incompatibility: Rh, ABO and minor blood groups) •RBC enzyme defects (e.g., G6PD deficiency, pyruvate kinase deficiency) •RBC structural abnormalities (hereditary spherocytosis, elliptocytosis) •Infection (sepsis, urinary tract infections) •Sequestered blood (e.g., cephalohematoma, bruising, intracranial hemorrhage) •Polycythemia •Shortened life span of fetal RBCs (80 vs. 120 d)
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2. Decreased hepatic uptake and conjugation of bilirubin
•Immature glucuronyltransferase activity in all newborns: term infants have 1% ofadult .activity, preterm infants have 0.1% •Gilbert Syndrome •CriglerNajjar Syndrome (Non-hemolytic Unconjugated Hyperbilirubinemia): inherited conjugation defect (very rare) •Pyloric stenosis (mechanism is unknown) •Hypothyroidism •Infants of Diabetic Mothers (polycythemia is also common) •Breastmilk Jaundice (pregnanediol inhibits glucuronyltransferase activity)
3. Increased enterohepatic reabsorption
•Breast feeding jaundice (due to dehydration from inadequate milk supply) •Bowel obstruction •No enteric feedings
CONJUGATED (DIRECT) HYPERBILIRUBINEMIA (CHOLESTASIS): Clinically, jaundice is green compared to jaundice due to unconjugated hyperbilirubinemia (yellow).
1. Hepatocellular diseases: A. Hepatitis: •Neonatal idiopathic hepatitis •Viral (Hepatitis B, C, TORCH infections) •Bacterial (E. coli, urinary tract infections) B. Total parenteral nutrition C. Hepatic ischemia (post-ischemic damage) D. Erythroblastosisfetalis (late, “Inspissated Bile Syndrome”) E. Metabolic disorders (partial list): •Glycogen storage disorders •Cerebrohepatorenal disease (Zellweger) •Cystic fibrosis •Hypopituitarism
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2. Biliary tree abnormalities:
A. Extrahepatic biliary atresia: In first 2 weeks,, unconjugated bilirubin Predominates; elevated conjugated bilirubin is late. B. Paucity of bile ducts (Alagille’s vs. non-syndromic)
Jaundice Yellow color of skin, nail beds & sclera caused by deposition of bilirubin secondary to increased bilirubin levels in blood (hyperbilirubinemia) Types of Jaundice•
1- Hemolytic Jaundice 2- Obstructive Jaundice 3- Hepatocellular Jaundice
Hemolytic Jaundice(Prehepatic)•
• Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis • Excess RBC lysis is commonly the result of autoimmune disease; hemolytic disease of the newborn (Rh- or ABO- incompatibility); structurally abnormal RBCs (Sickle cell disease); or breakdown of extravasated blood • High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL) Blood: Increased blood unconjugated (indirect) bilirubin Urine: Urobilinogen is increased No bilirubin in urine (Color of urine is normal) as it is bound to albumin Stool Dark color Increased stercobilin (produced from increased urobilinogen)
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Obstructive Jaundice(Posthepatic)•
In bile duct obstruction: Caused by an obstruction of the biliary tree
Conjugated bilirubin is prevented from passing to the intestine.
Thus, it is regarded to blood increasing conjugated (direct) bilirubin in blood
Excessive conjugated bilirubin is excreted in urine giving the yellowish
Brown color of urine
Blood: Increased conjugated (direct) bilirubin GGT & ALP are markedly elevated (ALT is normal or mildly elevated) Urine: Bilirubin appears in urine Thus, color is yellowish brown Urobilinogen is reduced Stool Pale (low stercobilin)
Hepatocellular Jaundice(Intrahepatic)•
First Liver damage (by hepatitis) causes low conjugation efficiency leading to increased unconjugated (indirect) bilirubin in blood Second Conjugated bilirubin is not efficiently secreted into bile. Instead, diffuses to blood increasing conjugated (direct) bilirubin in blood
Blood Increased BOTH unconjugated (indirect) & conjugated (direct) bilirubin ALT & AST levels are markedly elevated
Urine: Bilirubin is present in urine So, urine color is yellowish brown
Stool Pale (low stercobilin)
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URINE BLOOD
CONJUGA UNCONJUG UROBILI BILIR TED ALT & GGT & ATED NOGEN UBIN BILIRUBI AST ALP BILIRUBIN N NORMAL N: 0.2 – 1 N:0 - 0.2 NORM NORMA TRACE NIL mg/dl mg/dl AL L
HEMOLYT INCREAS INCREASE N:0 - 0.2 NORMA IC NIL NORM D D mg/dl L JAUNDICE AL
OBSTRUC Normal MARKE T. Decreased PRES N:0 - 0.2 INCREASE or D JAUNDICE or absent ENT mg/dl D mild INCREA increase SE
HEPATOC MARK Normal EL. Decreased PRES INCREASE INCREASE ED or JAUNDICE or absent ENT D D INCRE mild ASED increase
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Jaundice in Newborns•
In newborns (especially premature), •
Bilirubin accumulates as the liver enzyme bilirubin glucuronyltransferase(responsible for conjugation of bilirubin) is low at birth. (The enzymes reach adult levels in about 4 weeks) Accordingly, unconjugated bilirubin is increased in blood. Elevated bilirubin in excess of the binding capacity of albumin can diffuse into basal ganglia & cause toxic encephalopathy (kernicterus)
Treatment •
Exposure of the newborn skin to blue fluorescent light which converts Bilirubin to more polar & hence water-soluble isomers These isomers can be excreted into bile without conjugation to glucuronic acid.
Mechanism of Neonatal Jaundice Increased production of bilirubin •
Decreased uptake •
Decreased conjugation •
Increased enterohepatic circulation •
Congenital hyperbilirubinemia•
Bilirubin is elevated in blood due to inherited defects in the bilirubin metabolic pathway Crigler-Najjar syndrome Low activity of glucoronyltransferase (conjugating enzyme) Rare Inherited l disease Severe hyperbilirubinemia in neonates (unconjugated bilirubin) Complicated by kernicterus & early death
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What is non –physiologic or pathologic jaundice?
Jaundice should be considered non- physiologic or pathologic: if it occurs less than 24 hours after birth, jaundicein the first 24 hours after birth is not normal, and the causes of the jaundice must be investigated If bilirubin levels rise at a rate of greater than 5.0 mg/dl per day If total bilirubin levels exceed 15 mg/dl in a full – term or 10 mg/dl in preterm infant If conjugated bilirubin concentration is very high If evidence of acute hemolysis exists If hyperbilirubinemia persists beyond 10 days in a full-term or 21 days in a preterm infant.
Urobilinogen
Urobilinogen is a colourless by-product of bilirubin reduction. It is formed in the intestines by bacterial action on bilirubin. About half of the urobilinogen formed is reabsorbed and taken up via the portal vein to the liver, enters circulation and is excreted by the kidney.
Increased amounts of bilirubin are formed in hemolysis, which generates increased urobilinogen in the gut. In liver disease (such as hepatitis), the intrahepatic urobilinogen cycle is inhibited also increasing urobilinogen levels. Urobilinogen is converted to the yellow pigmented urobilin apparent in urine.
The urobilinogen in the intestine is directly reduced to brown stercobilin, which gives the feces their characteristic color. It can also be reduced to stercobilinogen, which can then be further oxidized to stercobilin. This constitutes the normal "enterohepatic urobilinogen cycle".
In biliary obstruction, below-normal amounts of conjugated bilirubin reach the intestine for conversion to urobilinogen. With limited urobilinogen available for reabsorption and
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Low urine urobilinogen may result from complete obstructive jaundice or treatment with broad-spectrum antibiotics, which destroy the intestinal bacterial flora. (Obstruction of bilirubin passage into the gut or failure of urobilinogen production in the gut.)
Low urine urobilinogen levels may result from congenital enzymatic jaundice (hyperbilirubinemia syndromes) or from treatment with drugs that acidify urine, such as ammonium chloride or ascorbic acid. Elevated levels may indicate hemolytic anaemia (excessive breakdown of red blood cells RBC), overburdening of the liver, increased urobilinogen production, re-absorption – a large hematoma, restricted liver function, hepatic infection, poisoning or liver cirrhosis.
What is fatty liver disease?
Fatty liver is a reversible condition wherein large vacuoles of triglyceride fat accumulate in liver cells via the process of steatosis (i.e., abnormal retention of lipids within a cell). Despite having multiple causes, fatty liver can be considered a single disease that occurs worldwide in those with excessive alcohol intake and the obese (with or without effects of insulin resistance). The condition is also associated with other diseases that influence fat metabolism. When this process of fat metabolism is disrupted, the fat can accumulate in the liver in excessive amounts, thus resulting in a fatty liver.
The accumulation of fat in alcoholic or non-alcoholic steatosis may also be accompanied by a progressive inflammation of the liver (hepatitis),
Causes
Fatty liver (FL) is commonly associated with alcohol or metabolic syndrome (diabetes, hypertension, obesity, and dyslipidemia), but can also be due to any one of many causes.
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