BIOCHEMISTRY OF LIVER

z CLASS № 33

THEME: BIOCHEMISTRY OF THE LIVER

1. Role of the liver in carbohydrate, lipid, amino acid and protein metabolism.

2. Detoxification functions of the liver.

3. Heme synthesis, reactions.

4. Degradation of heme. Bilirubin metabolism, scheme.

5. Disorders in bilirubin metabolism: jaundice, its types.

6. Biochemical mechanisms of hepatic failure and hepatic coma. Biochemical tests for diagnosis of liver disorders.

 

Liver is…

• The largest organ in the body

• Weighs 1.2 to 2 kg

• Constitutes about 2-3% of body weight.

• Involved in many digestive, vascular, and metabolic activities.

• Has over 500 vital functions.

MAJOR FUNCTIONS OF THE LIVER

I. Homeostatic function.

–        processing all of the gastrointestinal blood through the portal vein.

–        regulation the blood levels of glucose, amino acids, and other nutrients taken from food.

II. Biosynthetic function.

Production and secretion of compounds for extrahepatic tissues (most blood proteins, coagulation factors, lipids, glucose, ketone bodies, etc.).

III. Storage function.

Place for storage of glycogen, Fe, trace elements, vitamins (retinol, A, D, K, folic acid, B12).

III. Protective (detoxification) function.

-         Transformation of harmful substances (such as ammonia and toxins) into less harmful compounds.

-         Metabolism of most hormones, and ingested drugs to water soluble products for excretion.

-         Metabolism of ethanol. Ex.: Kupffer cells in the liver ingest bacteria or other foreign material from the blood.

V. Digestive function.

Synthesis of bile acids and production/secretion of the bile.

VI. Excretory function.

Excretion of various substances with the bile (water, cholesterol, bile pigments, phospholipids, bicarbonate and other ions).

VII. Metabolic function.

The central role in metabolism of most nutrients taken from food (carbohydrates, lipids, proteins, amino acids, porphyrins, etc.)

 

1. Role of the liver in carbohydrate, lipid, amino acid and protein metabolism.

01. Carbohydrate metabolism in the liver

Regulation of the blood glucose level.

Oxidative degradation of glucose either to CO2 and H2O, or lactate,

Synthesis of glycogen, conversion of glycogen to glucose,

Gluconeogenesis, or synthesis of glucose from non-carbohydrate compounds,

Conversion of glucose via pentose phosphate pathway,

Conversion of dietary monosaccharides, e.g. fructose and galactose to glucose.

Metabolism of glucose to glucoronic acid.

Role of the liver in the Cori Cycle

1. During intensive exercises skeletal muscles produce lactate from the glucose taken from the blood (anaerobic glycolysis).

2. The lactate is taken by the liver and converted back to glucose (gluconeogenesis)

3. The glucose is taken back to the muscles and used for production of energy

On average, the adult liver stores about 80 g of glycogen, and  in the fasting state releases 9 g of glucose each hour to the blood to maintain the peripheral glucose concentration.

The contribution from gluconeogenesis increases progressively with fasting as glycogen stores become further depleted at the rate of 11% per hour.

The carbon substrates for gluconeogenesis are derived from both lactate released by glycolysis in the peripheral tissues and from hepatic deamination of amino acids from the proteolysis of skeletal muscle. 

Energy for gluconeogenesis comes from the β-oxidation of fatty acids.

The end product of this process, acetyl-CoA, also stimulates the activity of the first committed enzyme of gluconeogenesis, pyruvate carboxylase.

02. Lipid metabolism in the liver

1) Uptake, b-oxidation of free fatty acids for energy.

2) Synthesis of triacylglycerols, phospholipids, cholesterol, and esters of cholesterol.

3) Metabolism of plasma lipoproteins (VLDL, HDL, chylomicron remnants).

4) Ketone body synthesis.

5) Metabolism of phospholipids.

6) Synthesis of bile acids.

7) Hydroxylation of the vitamin D.

Lipoprotein metabolism in the liver

• Triacylglycerols, phospholipids and cholesterol produced in the liver are packed into lipoproteins VLDL and HDL.

• The liver is the place for synthesis of apoproteins and enzymes for lipoprotein metabolism.

• Destruction of cholesterol-rich HDL and chylomicron “remnants”.

The liver synthesizes fatty acids from acetate units.

The fatty acids formed are then used to synthesize fats and phospholipids, which are released into the blood in the form of lipoproteins.

The liver’s special ability to convert fatty acids into ketone bodies and to release these again is also important.

The liver is the major site of both synthesis and catabolism of cholesterol, which is transported to other tissues as a component of lipoproteins.

Excess cholesterol is converted into bile acids in the liver or directly excreted with the bile. Bile acids are key elements in fat metabolism.

Bile acids have a detergent-like effect, solubilizing biliary lipids and emulsifying dietary fat in the gut to facilitate its digestion.

They are synthesized by hepatocytes.

03. Protein metabolism in the liver

1. Synthesis of proteins and enzymes for own use.

2. Synthesis of apo-proteins and enzymes for lipoprotein metabolism.

3. Production and secretion of the most of plasma proteins:

–        albumin,

–        ceruloplasmin,

–        transferrin,

–        clotting factors (fibrinogen, prothrombin, coagulation factors V, VII, IX, X, and XI),

–        acute-phase proteins (C-reactive protein, haptoglobin, etc.).

Amino acid metabolism in the liver

1) Maintenance of the plasma amino acid pool.

2) Catabolism of amino acids taken from blood stream: (deamination, transamination, transdeamination, etc.).

3) Synthesis of urea, detoxification of ammonia (convertion of α-ketoglutarate into glutamate).

4) Synthesis of creatine.

5) Formation of uric acid from purine bases.

2. Detoxification functions of the liver.

DETOXIFICATION and DRUG METABOLISM

• Detoxification reactions include conversion of toxic, nonpolar compounds to the less toxic and more readily extractable compounds.

• In detoxification the toxicity may be either completely eliminated, or lessened.

• The liver metabolizes most of

–        exogenous substances (xenobiotics, drugs, ethanol),

–      endogenous substances (steroid hormones, bilirubin, etc.).

Phase of detoxification in the liver

• Phase I:

involves a super family of CYP monooxygenases.

In these reactions the substance polarity increases by hydroxylation catalyzed by microsomal cytochrome P450 oxidases (microsomal oxidation).

• Phase II:

cytoplasmic enzymes conjugate the functional groups introduced in the first phase reactions, by glucuronidation, or other reactions.

Microsomal oxidation

• takes place in the endoplasmic reticulum (microsomes),

• conducts to hydroxylation of a non-polar substance R-H into the polar water-soluble product R-OH.

The process requires O2, NADPH(H+), a flavoprotein enzyme, and cytochrome P450.

R-H + O2 + NADPH + H+ → R-ОH + H2O + NADP+

Conjugation reactions

I. Glucuronidation.

Involves conjugation of a substance with glucuronic acid using UDP-glucuronic acid and a family of UDP-glucuronyl transferases.

• Ex. - Production of direct bilirubin from indirect bilirubin).

2. Conjugation with glycine

• Glycine is conjugated with such compounds as benzoic acid, nicotinic acid, para-aminobenzoic acid, etc.

• Hippuric acid is produced after introduction of sodium benzoate to the body.

The rate of synthesis and renal excretion of hippuric acid is measured to test the detoxification ability of the liver.

Sulfatation

• involves a sulfotransferase enzyme catalyzing the transfer of a sulfo group (-SO3 ) from 3'- phosphoadenosine-5'-phosphosulfate (PAPS), to a substrate molecule's hydroxyl or amine.

• Ex.: detoxification of indole and skatole that are toxic derivatives of tryptophan formed in bacterial putrefaction.

Other detoxification reactions

01. Acetylation reactions are used in detoxification of xenobiotics, and sulfonamide drugs.

X + AcetylCoA = Acetyl-X + CoA (where X is a xenobiotic)

02. Methylation reactions. (methylation of xenobiotics, pyridine and nicotinic acid with use of S-adenosine methionine).

 

3. Heme synthesis, reactions.

The heme moieties represent the prosthetic groups of hemoproteins:

–        hemoglobin, myoglobin,

–        cytochromes, oxygenases,

–        catalases.

• Synthesis of heme mainly takes place in

–        bone marrow (80 – 85%)

–        liver (15%).

• 300 mg of heme is produced daily in the body, of which only 1% is excreted unused in the urine and stools.

• 1/3 of the heme produced in the liver is required for the formation of cytochrome P 450.

• Heme synthesis begins in the mitochondria with condensation of succinyl-CoA with glycine to form δaminolaevulinic acid. 

The reaction in catalyzed by δaminolaevulinic acid synthase, which is the most crucial enzyme.

• After that a series of cytosolic reactions take place before being completed again in the mitochondria.

4. Degradation of heme. Bilirubin metabolism, scheme.

BILIRUBIN METABOLISM

• Bilirubin is the orange-yellow pigment derived from senescent red blood cells;

• It is a toxic waste product in the body;

• It is extracted and biotransformed mainly in the liver, and excreted in the bile and urine;

• It is a bile pigment;

• Elevations in serum and urine bilirubin levels are normally associated with Jaundice.

• Determination of the levels of total bilirubin, indirect and direct bilirubin, bile pigments is used for diagnosis of liver diseases.

• Metabolism of bilirubin takes place in the cells of reticuloendotelial system, the liver and intestine.

Formation of indirect bilirubin in the reticuloendothelial system

Indirect bilirubin

• In RES cells the ring structure of the heme is opened and the iron atom is removed by the action of heme oxygenase and cytochrome P450 to produce the green-colored intermediate biliverdin.

• Biliverdin is reduced by biliverdin reductase to form indirect bilirubin.

• Indirect (or unconjugated) bilirubin is a toxic, water-insoluble substance.

It is bound to serum albumin and transported to the sinusoidal membrane of the liver cells as a bilirubinalbumin complex.

Conjugation of indirect bilirubin in the liver

• In the liver, bilirubin is conjugated to two molecules of glucuronic acid to form bilirubin diglucuronide (or direct, conjugated bilirubin).

• Conjugated bilirubin is water-soluble, less toxic substance, which is subsequently eliminated via the bile and urine.

Conjugated form of bilirubin is normally present in the blood in 3 – 10% of the total serum bilirubin.

• The liver secretes conjugated bilirubin into the biliary canaliculi and finally to the small intestine

Conversion of bilirubin to other bile pigments

i.            In the intestine, conjugated bilirubin is converted back to unconjugated bilirubin by bacterial β-glucuronidases in the distal ileum and colon;

ii.            After that unconjugated bilirubin is reduced to the colored bile pigments: mesobilinogen, urobilinogen, and stercobilinogen.

iii.            Up to 80% of urobilinogen produced daily is reduced to stercobilinogen.

iv.            The rest 20% of urobilinogen is reabsorbed from the intestine and enters the enterohepatic circulation. The liver breaks down about 5 % of this urobilinogen to di- and tri-pyrrole compounds, which are excreted in the urine. The rest of the reabsorbed urobilinogen comes back to the intestine with the bile.

v.            In the colon, the stercobilinogen spontaneously oxidized to stercobilin (otherwise known as fecal urobilin), which is colored; most stercobilin is excreted in the feces, and is responsible for the color of feces.

vi.            A small fraction of stercobilinogen, (2 % – 5 %) enters the general circulation and appears in the urine.

Reference ranges of bile pigments in biological fluids

–        Total bilirubin – 5.0 – 20.5 mkmol/l (blood);

–        Indirect bilirubin – 1.7 – 17.1 mkmol/l (blood);

–        Direct bilirubin – 1.0 – 7.5 mkmol/l (blood);

–        Stercobilinogen – 4 mg/day (urine);

–        Stercobilin – 250 mg/day (feces).

• Normal urine does not contain bilirubin and urobilinogen

5. Disorders in bilirubin metabolism: jaundice, its types.

Jaundice

• Hyperbilirubinemia is elevated level of total bilirubin, resulted from imbalance between its production and excretion.

• Jaundice becomes clinically evident when the serum bilirubin level exceeds 27 - 34 mkmol/l.

Symptoms:

–        Icterus, or yellow discoloration of the skin, sclerae, and mucous membrane.

–        Itching due to deposits of bile salts on the skin;

–        Changes in the color of stool.

–        Deep orange and foamy urine.

Different causes of jaundice

–        Excessive Production of Bilirubin

–        Reduced Hepatocyte Uptake

–        Impaired Bilirubin conjugation

–        Impaired Bile Flow

Classification

I. Prehepatic (hemolytic)

II. Intrahepatic (hepatocellular)

III. Posthepatic (obstructive)

Prehepatic (hemolytic) Jaundice

• Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis.

Blood:

–        Increased indirect bilirubin

–        Unchanged direct bilirubin.

Intestine:

–        overproduction of urobilinogen and stercobilinogen from unconjugated bilirubin.

Urine:

–        increased level of stercobilinogen.

Stool:

–     Dark brown stool, markedly increased stercobilin.

Intrahepatic (hepatocellular) jaundice

• impaired hepatic uptake, conjugation, or secretion of bilirubin

• reflects low conjugation efficiency of hepatocytes resulted from liver disease, either inherited or acquired (hepatitis, etc.) .

Blood: – both indirect and direct bilirubin increased

Intestine: – Conjugated bilirubin is not efficiently secreted into the bile. Low production of stercobilinogen.

Urine: – Deep yellow because of excreted direct bilirubin. – Appearance of urobilinogen (impaired reduction of urobilinogen to the di- and tri-pyrrol end products).

Stool: – Reduced stercobilin, Pale coloured stool

Posthepatic (obstructive) jaundice

• Results from obstruction of the bile flow between the liver and intestine caused by structural disorders of the bile duct, tumors in the bile duct, cholelithiasis.

Blood: – Increased direct bilirubin – Unchanged indirect bilirubin.

Intestine: – Very low production of urobilinogen and stercobilinogen.

Urine: – Dark colored urine because of excretion of direct bilirubin.

Stool: – Clay colored stool. Absence of stercobilin.

High bilirubin in neonates

• Neonates are especially vulnerable to high unconjugated bilirubin levels due to an immature blood-brain barrier that predisposes them to kernicterus/bilirubin encephalopathy (bilirubin accumulates particularly in the basal nuclei),

which can result in permanent neurological damage with seizures, abnormal reflexes and eye movements etc.

• Neonates also have a low amount of functional UDP-glucuronyl-transferase and can have elevated unconjugated bilirubin, since conjugated is limited.

• Neonates in general are at increased risk since they lack the intestinal bacteria that facilitate the breakdown and excretion of conjugated bilirubin in the feces (this is largely why the feces of a neonate are paler than those of an adult).

Instead the conjugated bilirubin is converted back into the unconjugated form by the enzyme β-glucuronidase (in the gut, this enzyme is located in the brush border of the lining intestinal cells) and a large proportion is reabsorbed through the enterohepatic circulation.

6. Biochemical mechanisms of hepatic failure and hepatic coma. Biochemical tests for diagnosis of liver disorders.

Hepatic failure and hepatic coma

• Liver failure or hepatic failure is the inability of the liver to perform its normal synthetic and metabolic function.

• Two forms are recognized, acute and chronic liver failure.

Acute liver failure

• Is the rapid development of hepatocellular dysfunction,

specifically coagulopathy and mental status changes (encephalopathy) in a patient without known prior liver disease.

• Hepatocellular disease may ALTER PROTEIN SYNTHESIS in the liver, especially plasma albumin, and coagulation factors II, VII, IX, and X.

• The diagnosis of acute liver failure is based on physical exam, laboratory findings, patient history, and past medical history to establish mental status changes, coagulopathy, rapidity of onset, and absence of known prior liver disease respectively

Chronic hepatic failure

• usually occurs in the cirrhosis as the result of many possible causes, such as excessive alcohol intake, hepatitis B or C, autoimmune, hereditary and metabolic causes (iron or copper overload, steatohepatitis or nonalcoholic fatty liver disease.

• Main causes of the liver failure:

–        Acute viral hepatitis;

–        Cirrhosis (alcoholic or non-alcoholic);

–        Excessive injuries or traumas;

–        Sepsis;

–        Poisonings by hepatotrophic venoms and medicines.

Clinical findings in liver failure

–        Hyperbilirubinemia;

–        Low total serum protein and albumin level;

–        Coagulopathy and hemorrhage because of impaired synthesis of clotting proteins;

–        Low levels of potassium, sodium, and calcium in the blood;

–      High levels of toxic phenol and indol derivatives, aromatic, branched and sulfur-containing amino acids in the blood.

Hepatic coma

is a state of unconsciousness which the patient cannot be aroused, even by powerful stimuli.

Hepatic coma accompanies cerebral damage resulting from degeneration of liver cells especially that associated with cirrhosis of the liver.

Liver functional tests

• are groups of blood tests that provide information about the state of a patient's liver.

A panel of biochemical measurements is routinely performed in the clinical laboratories on plasma or serum specimens.

• The standard liver panel includes determination of:

–        Total serum protein and albumin (low plasma albumin is detected in acute and chronic liver diseases);

–        Total bilirubin, direct and indirect bilirubin, other bile pigments;

–        Blood ammonia (elevated in cirrhosis of the liver and disorders of the urea cycle);

–        Alanine aminotransferase (AlAT), aspartate aminotransferase (AsAT) (higher increases in the AlAT activity compared to the AsAT activity);

–        Alkaline phosphatase (ALP) (increases in cholestasis);

–      Gamma-glutamyl transferase (GTT) (increases in alcohol abuse and hepatitis).

Other liver tests

Coagulation test.

–        prothrombin time

–        prothrombin ratio (PR)

–        international normalized ration (INR);

Determination of ceruloplasmin, serum glucose, cholesterol, urea.

Determination of alpha-fetoprotein (AFP) (increases in hepatocelular carcinoma)

Lactate dehydrogenase (LDH4 and LDH5)