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Transport of Bilirubin

Transport of Bilirubin


Transport  of Bilirubin

  • Unconjugated bilirubin (UCB), the principal mammalian bile pigment, is the end intravascular product of heme catabolism.
  • Bilirubin is toxic to tissues; therefore, it is transported in the blood bound to albumin.
  • Bilirubin in the bloodstream is usually in a free, or unconjugated, state.
  • Bilirubin leaves the site of production in the reticuloendothelial system and is transported in plasma bound to albumin .
  • The capacity of serum albumin to bind bilirubin is known as the binding capacity, and the strength of the bilirubin-albumin bond is referred to as the binding affinity.
  • In  100 ml of plasma,  approximately 25  mg of  bilirubin can  be  tightly  bound  to  albumin  at its  high-affinity  site.
  • The conjugation  of bilirubin  is  catalyzed  by a specific enzyme called glucuronyltransferase.
  • proteins Ligandin  (a member  of  the  family  of  glutathione S-transferases) and Protein Y help in  intracellular  binding
  • In  the  liver  the  bilirubin is  removed  from albumin, Taken  up at the  sinusoidal surface  of the  hepatocytes by  a carrier-mediated  saturable system  (facilitated transport  system). It is then concentrated to about 1,000 times the strength found in blood plasma.
  • Much bilirubin leaves the liver and passes to the gallbladder, where it is further concentrated and mixed with the other constituents of bile. 
  • conjugated bilirubin passes from the gallbladder or liver into the intestine. There, it is reduced by bacteria to mesobilirubinogen and urobilinogen.
  • Some urobilinogen is reabsorbed back into the blood; the rest goes back to the liver or is excreted from the body in urine and fecal matter. In humans, bilirubin is believed to be unconjugated until it reaches the liver.
  • 80-90%  of the  urobilinogen stercobilinogen  and  stercobilin and  excreted  through  feces.
  • 70-20 %  enterohepatic  circulation reaches  the  liver, This  is  called  enterohepatic urobilinogen  cycle.
  • A small  fraction  < 3 mg/dl escape  hepatic  uptake, filters  across  renal  glomerulus  and  is  excreted through  urine.
Don’t Forget to Solve all the previous Year Question asked on Transport of Bilirubin

Module Below Start Quiz

Transport of Bilirubin

Transport of Bilirubin

Q. 1 Bilirubin is absent in urine because it is :

 A

Distributed in the body fat

 B

Conjugated with glucoronide

 C

Not filterable

 D

Lipophilic.

Q. 1

Bilirubin is absent in urine because it is :

 A

Distributed in the body fat

 B

Conjugated with glucoronide

 C

Not filterable

 D

Lipophilic.

Ans. D

Explanation:

Unconjugated serum bilirubin is always bound to albumin, is not filtered by the kidney (not found in the urine)
In biliary obstruction or hepatocellular diseases, both conjugated and unconjugated bilirubin accumulate in plasma.
In hemolytic jaundice, total plasma bilirubin increases, but the proportion of the unconjugated and conjugated fractions remains unchanged.


Q. 2

Bilirubin is secreted by:

 A

Bile Salts

 B

Bile pigments

 C

Secretin

 D

CCK.

Q. 2

Bilirubin is secreted by:

 A

Bile Salts

 B

Bile pigments

 C

Secretin

 D

CCK.

Ans. A

Explanation:

A i.e. Bile salts

Substances that increase the secretion of bile are called as cholerecticsQ. Bile salts are amongst the most important physiological cholerectionQ


Q. 3 Bilirubin is the degradation product of –

 A

Albumin

 B

Globulin

 C

Heme

 D

Transferrin

Q. 3

Bilirubin is the degradation product of –

 A

Albumin

 B

Globulin

 C

Heme

 D

Transferrin

Ans. C

Explanation:

Ans. is ‘c’ i.e., Heme

Bilirubin metabolism

o Bilirubin is the end product of heme degradation.

o The heme is derived from –

(i)       Senescent erythrocytes by mononuclear phagocytic system in the spleen, liver and bone marrow (major source).

(ii)     Turnover of hemoproteins (e.g. cytochrome p.450).

o Heme is oxidized to biliverdin by heme oxygenase.

o Biliverdin is then reduced to bilirubin by biliverdin reductase.

o Bilirubin is transported to liver in bound form with albumin.

o There is carrier mediated uptake of bilirubin in the liver.

o This bilirubin is conjugated with glucuronic acid by UDP glucuronyl transferase (UGT1A1) to from conjugated bilirubin (bilirubin glucronides).

o Conjugated bilirubin is excreted into bile.

o Most of the conjugated bilirubin is deconjugated and degraded to urobilinogen.

o The most of the urobilinogen is excreted in the feces.

o Approximately 20% of the urobilinogen is reabsorbed in the ileum and colon and is returned to the liver, and promptly rexcreted into bile —> Enterohepatic circulation.

o The small amount that escapes this enterohepatic circulation is excreted in urine.

Quiz In Between


Q. 4 Bilirubin is absent in urine because it is‑

 A

Distributed in the body fat

 B

Conjugated with glucoron

 C

Not filtered

 D

None

Q. 4

Bilirubin is absent in urine because it is‑

 A

Distributed in the body fat

 B

Conjugated with glucoron

 C

Not filtered

 D

None

Ans. C

Explanation:

Answer is C (Not filtered)

Normal urine does not contain bilirubin because normal blood contains bilirubin in the uncongugated form. Unconjugated bilirubin is lipid soluble or lipophilic (water insoluble) because it is transported in the blood as a complex with albumin (albumin-bilirubin complex) which is not allowed to filter through the glomerulus.

Although conjugated bilirubin is water soluble and filterable at the glomurulus, conjugated bilirubin is not present in the blood normally and hence does not filter to appear in the urine.

Unconjugated bilirubin present in blood is complexed with albumin to make it soluble in blood and transport it to the liver. However, the glomerulus does not allow the albumin bilirubin complex to filter and hence bilirubin does not appear in urine.

Conjugated bilirubin is formed in the liver and directly excreted into the GIT through bile where it is reduced to urobilinogen and stercobilinogen. Conjugated bilirubin does not normally circulate in the blood at all and hence despite being filterable and water soluble it does not appear in urine.


Q. 5

Urobilinogen is formed in the:   
September 2006

 A

Liver

 B

Kidney

 C

Intestine

 D

Spleen

Q. 5

Urobilinogen is formed in the:   
September 2006

 A

Liver

 B

Kidney

 C

Intestine

 D

Spleen

Ans. C

Explanation:

Ans. C: Intestine

Urobilinogen is a colourless product of bilirubin reduction. It is formed in the intestines by bacterial action. Some urobilinogen is reabsorbed, taken up into the circulation and excreted by the kidney. This constitutes the normal “enterohepatic urobilinogen cycle”.

Urobilinogen content is determined by a reaction with Ehrlich’s reagent, which contains para-Dimethyl amino benzaldehyde and may be measured in Ehrlich units

Quiz In Between


Q. 6 Bilirubin bound inside hepatocyte to ‑

 A

Albumin

 B

Ubiquinone

 C

Ligandin

 D

Globulin

Q. 6

Bilirubin bound inside hepatocyte to ‑

 A

Albumin

 B

Ubiquinone

 C

Ligandin

 D

Globulin

Ans. C

Explanation:

Ans. is ‘c’ i.e., Ligandin

Bilirubin metabolism

Bilirubin is the end product of heme degradation.

The heme is derived from –

i) Senescent erythrocytes by mononuclear phagocytic system in the spleen, liver and bone marrow (major source).

ii) Turnover of hemoproteins (e.g. cytochrome p.450).

Heme is oxidized to biliverdin by heme oxygenase.

Biliverdin is then reduced to bilirubin by biliverdin reductase.

Bilirubin is transported to liver in bound form with albumin.

Bilirubin is transferred to hepatocytes where it is bound to ligandin.

There is carrier mediated uptake of bilirubin in the liver.

This bilirubin is conjugated with glucuronic acid by UDP glucuronyl transferase (UGT1A1) to from conjugated bilirubin (bilirubin glucronides).

Conjugated bilirubin is excreted into bile.

Most of the conjugated bilirubin is deconjugated and degraded to urobilinogen.

The most of the urobilinogen is excreted in the feces.


Q. 7 All are involved in bilirubin metabolism except‑

 A

ALA synthase

 B

Heme oxygenase

 C

Biliverdine reductase

 D

Glucuronyl transferase

Q. 7

All are involved in bilirubin metabolism except‑

 A

ALA synthase

 B

Heme oxygenase

 C

Biliverdine reductase

 D

Glucuronyl transferase

Ans. A

Explanation:

Ans. is ‘a’ i.e., ALA synthase 

Quiz In Between



Hyperbilirubinemias

hyperbilirubinemia

Q. 1

Following are causes of unconjugated hyperbilirubinemia, except:
 A Hemolytic anemia

 B

Large hematoma

 C

Rotor syndrome

 D

Megaloblastic anemia

Q. 1

Following are causes of unconjugated hyperbilirubinemia, except:

 A Hemolytic anemia

 B

Large hematoma

 C

Rotor syndrome

 D

Megaloblastic anemia

Ans. C

Explanation:

Answer is C (Rotor syndrome)
Rotor’s syndrome is an Autosomal recessive inherited disorder characterized by a deject in biliary excretion leading to conjugated hyperbilirubinemia:

Indirect hyperbilirubinemia                                                              

Direct hyperbilirubinemia

A.   Hemolytic disorders

A.   Inherited conditions

1.    Inherited

1.     Dubin-Johnson syndrome

a.   Sperocyteosis, elliptocytosis

2.     Rotor’s syndrome

Glucose-6-phosphate dehydrogenase and pyruvate kinase deficiencies

b. Sickle cell anemia

 

2.    Acquired                         _

a. Microangiopathic hemolytic anemias

b. Paraoxysmal nocturnal hemoglobinuria

c. Immune hemolysis

 

B.    Ineffective erythropoesis

 

1.    Cobalamin, folate, thalassemia, and severe iron deficiencies

 

C. Drugs

 

1.    Rifampicin, probenbecid, ribavirin

 

D.   Inherited conditions

 

1.    Crigler-Najjar types I and II

 

2.    Glibert’s syndrome

 


Q. 2 Conjugated hyperbilirubinemia is seen in all EX­CEPT:
March 2013

 A

Dubin Johnson syndrome

 B

Rotor syndrome

 C

Gilbert syndrome

 D

None of the above

Q. 2

Conjugated hyperbilirubinemia is seen in all EX­CEPT:
March 2013

 A

Dubin Johnson syndrome

 B

Rotor syndrome

 C

Gilbert syndrome

 D

None of the above

Ans. C

Explanation:

Ans. C i.e. Gilbert syndrome
Gilbert syndrome presents with unconjugated hyperbilirubinemia


Q. 3

Unconjugated hyperbilirubinemia is seen in all of the following except:   
March 2010

 A

Crigler Najjar Syndrome

 B

Physiological jaundice

 C

Dubin-Johnson syndrome

 D

Gilbert syndrome

Q. 3

Unconjugated hyperbilirubinemia is seen in all of the following except:   
March 2010

 A

Crigler Najjar Syndrome

 B

Physiological jaundice

 C

Dubin-Johnson syndrome

 D

Gilbert syndrome

Ans. C

Explanation:

Ans. C: Dubin-Johnson Syndrome

Dubin-Johnson syndrome is an autosomal recessive disorder that causes an increase of conjugated bilirubin without elevation of liver enzymes (ALT, AST).

This condition is associated with a defect in the ability of hepatocytes to secrete conjugated bilirubin into the bile.

The conjugated hyperbilirubinemia is a result of defective endogenous and exogenous transfer of anionic conjugates from hepatocytes into the bile.

Pigment deposition in lysosomes causes the liver to turn black.

Other causes of conjugated/direct hyperbilirubinemia:

  • Hepatocellular diseases:

– Hepatitis:

  • Neonatal idiopathic hepatitis
  • Viral (Hepatitis B, C, TORCH infections)
  • Bacterial (E. colt, urinary tract infections)

–        Total parenteral nutrition

–        Hepatic ischemia (post-ischemic damage)

–        Erythroblastosis fetalis (late, “Inspissated Bile Syndrome”)

Metabolic disorders:

  • Alpha-1 antitrypsin deficiency
  • Galactosemia, tyrosinemia, fructosemia
  • Glycogen storage disorders
  • Cystic fibrosis

Biliary tree abnormalities:

–         Extrahepatic biliary atresia: In first 2 weeks, unconjugated bilirubin predominates; elevated conjugated bilirubin is late.

–        Paucity of bile ducts

–        Choledochal cyst

–        Bile plug syndrome

Causes of unconjugated/indirect hyperbilirubinemia:

  • 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)

–        Neonatal Jaundice

–        Polycythemia

–        Shortened life span of fetal RBCs

Decreased hepatic uptake and conjugation of bilirubin

–        Immature glucuronyl transferase activity in all newborns: term infants have 1% of adult activity, preterm infants have 0.1%.

–        Gilbert Syndrome

–        Crigler Najjar Syndrome (Non-hemolytic Unconjugated Hyperbilirubinemia): inherited conjugation defect (very rare)

–        Breastmilk Jaundice (pregnanediol inhibits glucuronyl transferase activity)

Increased enterohepatic reabsorption

–        Breastfeeding jaundice (due to dehydration from inadequate milk supply)

Quiz In Between


Q. 4 Conjugated hyperbilirubinemia

 A

Dubin johnson syndrome

 B

Criggler naj jar syndrome

 C

Breast milk jandice

 D

Gilbert syndrome

Q. 4

Conjugated hyperbilirubinemia

 A

Dubin johnson syndrome

 B

Criggler naj jar syndrome

 C

Breast milk jandice

 D

Gilbert syndrome

Ans. A

Explanation:

Ans. is ‘a’ i.e., Dubin johnson syndrome
Breast milk jaundice –

  • Decrease bilirubin uptake across hepathocyte membrane.
  • Entero-hepatic recirculation.
  • Leads to indirect hyperbilirubinemia.

Crigler naj jar & Gilbert syndrome (deficiency of glucuronyl transferase)

  • Decrease conjugation leads to Indirect hyperbilirubinemia.
  • Defect in hepatocyte secretion of conjugated bilirubin.
  • Leads to direct hyperbilirubinemia

Q. 5 Causes of unconjugated hyperbilirubinemia include?

 A

Sepsis

 B

Criggler-Najar syndrome

 C

Rotor syndrome

 D

Gilbert syndrome

Q. 5

Causes of unconjugated hyperbilirubinemia include?

 A

Sepsis

 B

Criggler-Najar syndrome

 C

Rotor syndrome

 D

Gilbert syndrome

Ans. A:B:D:E

Explanation:

Answer- A, B, D, E, Sepsis, Criggler-Najar syndrome, Gilbert syndrome, Intravascular hemolysis
Unconjugated hyperbilirubinemia:-

  • Increased production of bilirubin from hemoglobin, So that the capacity of liver to conjugate bilirubin is overwhelmed by increased production, e.g.
  1. Hemolytic anemia (both intravascular and extamascular)s Hereditary sphnocytosis, G6PD defciency.
  2. Inefrective erythropoiesis- Thalassemia, Pernicious anemia.
  3. Reduced hepatic uptake of bilirubin from bilirubin – albumin complex > Drugs,
  4. Infections:- Sepsis, UTI
  5. Impaired hepatic conjugation.

Q. 6 Congenital hyperbilirubinemia is/are seen in: 

 A

Prematurity

 B

Hypoalbuminaemic state

 C

Hepatitis

 D

Sepsis

Q. 6

Congenital hyperbilirubinemia is/are seen in: 

 A

Prematurity

 B

Hypoalbuminaemic state

 C

Hepatitis

 D

Sepsis

Ans. A:C:D:E

Explanation:

Ans. a. Prematurity; c. Hepatitis; d. Sepsis; e. Polycythemia
Albumin less than 3.0 mg/dl is risk for hyperbilirubinemia neurotoxicity
Two other groups of disorders are associated with hyperbilirubinemia:
(1) Unconjugated hyperbilirubinemia seen in,

  • Breast milk jaundice
  • Blood group incompatibility
  • Lucey-Driscoll syndrome
  • Congenital hypothyroidism
  • Upper intestinal obstruction
  • Gilbert disease
  • Crigler-Najjar syndrome
  • Hereditary spherocytosis
  • Non-spherocytic hemolytic anemia
  • Drug-induced hyperbilirubinemia

(2) Conjugated hyperbilirubinemia present in,

  • Dubin-Johnson syndrome
  • Rotor syndrome
  • Biliary atresia
  • Neonatal hepatitis

Quiz In Between



Hyperbilirubinemias

HYPERBILIRUBINEMIAS


HYPERBILIRUBINEMIAS

Depending on the nature of the bilirubin elevated, the condition may be grouped into:-

→ conjugated or Unconjugated  hyperbilirubinemia.

A → Congenital  Hyperbilirubinemias

They result from abnormal uptake, conjugation or excretion of bilirubin due to inherited defects such as

Crigler-Najjar Syndrome

  • The defect is due to conjugation, there is severe deficiency of UDP glucuronyl  transferase. The disease is often fatal and the children die before the age of 2.
  • Unconjugated bilirubin level increases to more than 20mg/dl, and hence kernicterus results.
  • Bilirubin level  in blood exceeds 20 mg/dl in Crigler-Najjar syndrome Type 1 and does not exceed 20 mg/dl in Crigler-Najjar syndrome Type 2.

Gilbert’s Disease:

  • It is inherited as an autosomal dominant trait.
  • The defect is in the uptake of bilirubin by the liver.
  • Bilirubin level is usually around 3 mg/dl, and patient is asymptomatic, except for the presence of mild jaundice.

Dubin-Johnson Syndrome:

  • It is an autosomal recessive trait leading to defective excretion of conjugated bilirubin.
  • The disease results from the defective ATP-dependent organic anion transportin bile canaliculi.
  • There is a mutation in the MRP-2 protein which is responsible for transport of  conjugated bilirubin into bile.
  • The bilirubin gets deposited in the liver and the liver appears black. The condition is referred to as Black liver jaundice.

Rotor Syndrome

  • exact defect is not identified. Bilirubin excretion is defective, but there is no staining of the liver. It is an autosomal recessive condition.

B→Acquired Hyperbilirubinemias:

Physiological Jaundice:

  • Called as neonatal hyperbilirubinemia.
  • Transient  hyperbilirubinemia  is due to an accelerated rate of destruction of RBCs and also because of the immature hepatic system of conjugation of bilirubin.

Breast milk jaundice

  • In some breast-fed infants, prolongation of the  jaundice has been attributed to high level of an estrogen derivative in maternal blood, which is excreted through the milk.

Conjugated Hyperbilirubinemias

  • Dubin Johnson’s syndrome
  • Rotor syndrome
  • Benign Recurrent intrahepatic Cholestatsis (BRIC)
  • Progressive Familial intrahepatic Cholestatsis (FIC)

Exam Important

  • Unconjugated Hyperbilirubinemia is associated with > 85% indirect bilrubin or less than 15% of direct bilirubin.
  • Hemolytic disorders and increased hemoglobin destruction cause unconjugated or indirect hyperbilirubinemia.
  • Biliary atresia and neonatal hepatitis lead to conjugated hyperbilirubinemia.
  • Uncojugated hyperbilirubinemias is-Gilbert’s disease, Crigler-Najjarsyndrome.
  • Conjugated Hyperbilirubinemias-A. Dubin Johnson’s syndrome, B. Rotor syndrome, c.Benign Recurrent intrahepatic Cholestatsis (BRIC), d.Progressive Familial intrahepatic Cholestatsis (FIC)
Don’t Forget to Solve all the previous Year Question asked on HYPERBILIRUBINEMIAS

Module Below Start Quiz

Polymerase Chain Reaction (Pcr)

POLYMERASE CHAIN REACTION (PCR)


POLYMERASE CHAIN REACTION (PCR)

  • Karry Mullis invented this ingenious method in 1989, & awarded Nobel prize in 1993.
  • millions of copies of a particular sequence of DNA can be produced within a few hours.
  • PCR  is  a method  of enzymatic  amplification  of a target  sequence  of DNA.
  • It is sensitive, selective (specific) and extremely rapidmeans of amplifiing any desired sequence of double stranded DNA.
  • DNA  to  be amplified  is  replicated  by DNA polymerase  of  Thermus  aquaticus  (Taq) because  it  is  thermostable.
  • Primers  are  amplified  to  produce  desired  sequence  of  DNA.
  • Two DNA primers of about 20-30 nucleotides with complementary sequence of the flanking region can be synthesized.

Steps  in  PCR:

  • PCR  uses DNA  polyme  rase.
  • Each  cycle  doubles  the  amout  of DNA  in  the  sample,  leading  to  exponential  increase.
  • Thus amplification after’n’number of cycle in (2)n, twenty cycles provide an amplification of 106 (million) and 30 cycles of 10, (billion).

Step 1: Separation (Denaturation):DNA strands are separated (melted) by heating at 95°C for 15 seconds to 2 minutes

Step 2: Priming (Annealing): The primers are annealed by cooling to 50°C for 0.5 to 2 minutes. The primers hybridise with their complementary single stranded DNA produced in the first step.

Step 3: Polymerization: New DNA strands are synthesized by Taq polymerase.This enzyme is derived from bacteria Thermus acquaticus that are found in hot springs. Therefore the enzyme is not denatured at high temperature. The polymerase reaction is allowed to take place at 72°C for 30 seconds in presence of dNTPs (all four deoxy ribonucleotide triphosphates) and DNA polymerase. 

  • As Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
  • Both strands of DNA are now duplicated 
  • The steps of 1,2 and 3 are repeated, Thus, 20 cycles provide for 1 million times amplifications. These cycles are generally repeated by auto-mated instrument, called Tempcycler.

Thus following is required in PCR:- 

  1. target double standard DNA,
  2. two specific primrers
  3. a thermostable DNA polymerase
  4. Taq polymerase,
  5. dNTP

APPLICATION OF PCR:

Detection of infectious diseases: 

  • AIDS, Tuberculosis, CMV, H1N1, etc.
  • Lyme Disease-joint inflammation from tick bites.
  • Detect 3 sexually transmitted diseases in one swab-herpes, papillomarvirus, chlamydia.
  • PCR can diagnosis even one bacteria or virus present in the specimen.
  • Latent viruses can also be diagnosed.

Detection of Variations and Mutations in Genes

  • Detects people with inherited disorders and carriers
  • Track presence or absence of DNA abnormalities, characteristic to cancer.
  • Prenatal diagnosis of genetic disorders.
  • PCR combined with RE and Southern blotting is used for mutation detection.

PCR and the Law

  • DNA fingerprinting can multiply amounts of DNA
  • found in blood samples, hair, semen, and other
  • body fluids “contain only very few tubercle bacilli ,cytomegalo virus and HIV

Various types  of PCR:

  1. Multipler PCR
  2. Reverse Tanscriptase PCR: DNA polymerase derived from thermus thermophillus organism has got additional reverse transcriptase activity and hence is preferred for reverse transcriptase type of PCR.
  3. Realtime PCR: a fluorescent dye known as “SYBR green is used to tag the primer, this helps in quantitative detection of PCR material.
  4. Invert  PCR
  5. Nested PCR.

Exam Important

  • PCR  is  a method  of enzymatic  amplification  of a target  sequence  of DNA.
  • It is sensitive, selective (specific) and extremely rapidmeans of amplifiing any desired sequence of double stranded DNA.
  • the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq). Taq polyrnerase is used because it is thermostable.
  • Each  cycle  doubles  the  amout  of DNA  in  the  sample,  leading  to  exponential  increase.
  • In Chain extension:- DNA polymerase and deoxyribonucleotides are added. to the mixture.
  • Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
  • DNA polymerase derived from thermus thermophillus organism has got additional reverse transcriptase activity and hence is preferred for rsverse transcriptase tnte of PCR.
Don’t Forget to Solve all the previous Year Question asked on POLYMERASE CHAIN REACTION (PCR)

Module Below Start Quiz

Polymerase Chain Reaction (Pcr)

PCR

Q. 1 PCR is done for:       
UP 11; UPSC 13; AIIMS 13

 A

Cloning of DNA in cells

 B

Replication of DNA in vitro

 C

Sequencing of DNA

 D

Both A and B

Q. 1

PCR is done for:       
UP 11; UPSC 13; AIIMS 13

 A

Cloning of DNA in cells

 B

Replication of DNA in vitro

 C

Sequencing of DNA

 D

Both A and B

Ans. B

Explanation:

Ans. Replication of DNA in vitro


Q. 2

PCR does not require:
AIIMS 07

 A

Primer

 B

DNA-fragments

 C

DNA polymerase

 D

Radio-labeled DNA probe

Q. 2

PCR does not require:
AIIMS 07

 A

Primer

 B

DNA-fragments

 C

DNA polymerase

 D

Radio-labeled DNA probe

Ans. D

Explanation:

Ans. Radio-labeled DNA probe


Q. 3

Not a component of PCR ‑

 A

Primer

 B

Taq polymerase

 C

DNA Polymerase

 D

Restriction enzyme

Q. 3

Not a component of PCR ‑

 A

Primer

 B

Taq polymerase

 C

DNA Polymerase

 D

Restriction enzyme

Ans. D

Explanation:

 

Steps in PCR

PCR uses DNA polymerase to repetitively amlify targeted portion of DNA. Each cycle doubles the amout of DNA in the sample, leading to exponential increase with repeated cycles of amplification. Thus amplification after ‘n’ number of cycle in (2)”. Twenty cycles provide an amplification of 106 (million) and 30 cycles of 109 (billion).

PCR occurs in following steps –

i)      Isolation of target DNA sequence :- 

ii)     Primers construction:- 

iii)    Denaturation of DNA :- 

iv)    Annealing of primers to single stranded DNA :- 

v)     Chain extension:- 

Thus following are required in PCR :- Target double stranded DNA, two specific primers, a thermostable DNA polymerase (Taq polymerase), deoxyribonucleotides (dNTP).

Quiz In Between


Q. 4 In PCR, DNA polymerase is derived from‑

 A

Experimental E coli

 B

Thermus aquaticus

 C

Retroviruses

 D

Bacteriophages

Q. 4

In PCR, DNA polymerase is derived from‑

 A

Experimental E coli

 B

Thermus aquaticus

 C

Retroviruses

 D

Bacteriophages

Ans. B

Explanation:

Ans. is ‘b’ i.e., Thermus acquaticus

PCR is a method of enzymatic amplification of a target sequence of DNA.

It is sensitive, selective (specific) and extremely rapid means of amplifying any desired sequence of double stranded DNA, which can be as short as 50-100 base pairs (bp) and as long as 10 kbp.

In PCR, the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq).

Taq polymerase is used because it is thermostable, not denatured at a temperature upto 95°C (in PCR DNA is to be heated to 94°-95° C for separation of strands).


Q. 5

Enzyme used in PCR is ‑

 A Reverse transcriptase 

 B

Tag polymerase

 C

RNA polymerase

 D

None

Q. 5

Enzyme used in PCR is ‑

 A

Reverse transcriptase 

 B

Tag polymerase

 C

RNA polymerase

 D

None

Ans. B

Explanation:

Ans. is ‘b i.e., Taq polymerase 

PCR is a method of enzymatic amplification of a target sequence of DNAe.

  • It is sensitive, selective (specific) and extremely rapid means of amplifying any desired sequence of double stranded DNAe, which can be as short as 50-100 base pairs (bp) and as long as 10 kbp.
  • In PCR, the DNA to be amplified is replicated by DNA polymerase of Thermus aquaticus (Taq). Taq polymerase is use because it is thermostable0, not denatured at a temperature upto 95°C (in PCR DNA is to be heated to 94°-95° C for separation of strands).
  • For amplifying a desired DNA sequence in DNA, we have to know short flanking sequences on either side of the target segue nce so that complementary primers can be prepared.
  • Primers0 are the synthetic oligonucleotides of 20-35 sequence, which have sequence complementary to flanking sequence, i.e. sequence of flanking region of target DNA sequence.
  • Primers are amplified to produce desired sequence of DNA.

Q. 6 Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Q. 6

Real Time PCR is used for:

 A

Multiplication of RNA

 B

Multiplication of specific segments of DNA

 C

Multiplication of Proteins

 D

To know how much amplification of DNA has occurred

Ans. D

Explanation:

Ans. (d) To know how much amplification of DNA has occured PF 1,m,etz. 25/e 714, 27/e, p 124; Greenwood 18/e 79

Real Time. PCR

  • It is the molecular detection technique that discriminates real time amplification from conventional PCR assays.
    • The real-time polymerase chain reaction (PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction.
    • This combines the DNA amplification and detection steps into one homogeneous assay and obviates the requirement for gel electrophoresis to detect amplification products.
    • Its simplicity, specificity, and sensitivity, together with its, more reliable instrumentation, and improved protocols, has made realtime                     oenLhmark technology for lite ocLuLtion of DNA.
    • Real time PCR is extremely useful in medical microbiology, with greatest impact on virology.

Quiz In Between


Q. 7 Which is not a step of PCR ‑

 A

Annealing

 B

Extension

 C

Transformation

 D

Denaturation

Q. 7

Which is not a step of PCR ‑

 A

Annealing

 B

Extension

 C

Transformation

 D

Denaturation

Ans. C

Explanation:

Ans. is ‘c’ i.e., Transformation [Ref Lippincott’ s 5thle p. 479-83; Harper 28th/e p. 395] Steps in PCR

  • Isolation of target DNA sequence→  Primer construction → Denaturation of DNA→ Annealing of primers to single stranded DNA→ Chain extension.

Q. 8 Which of the following enzymes have proof reading function in PCR [Polymerase Chain Reaction] 

 A

Taq polymerase

 B

PFU Polymerase

 C

Thermos thermophilus

 D

Thermal flavus (Replinase)

Q. 8

Which of the following enzymes have proof reading function in PCR [Polymerase Chain Reaction] 

 A

Taq polymerase

 B

PFU Polymerase

 C

Thermos thermophilus

 D

Thermal flavus (Replinase)

Ans. B:E

Explanation:

Ans. is ‘b’ i.e., PFU Polymerase; & ‘e’ i.e., T-7 polymerase [Ref Textbook of PCR by Mike McPherson]

  • The use of high fidelity DNA polymerases in PCR is essential for reducing the introduction of amplification errors in PCR products.
  • Several thermostable DNA polymerases with 3′ → 5′ exonuclease – dependent proofreading activity have been introduced for high.
  • Pfu DNA polymerase → Derived from Pyrococcus fusarious.
  • Pwo DNA polymerase → Isolated from Pyrococcus woesei.
  • KOD HiFi DNA polymerase → Isolated from Thermococcus Kodakaraensis.
  • T7 DNA polymerase.

Q. 9

Which of the following is/are true about PCR except:

 A

Uses heat labile DNA polymerase

 B Uses heat stable DNA polymerase

 C

Is technique for DNA amplification

 D

Used to yield multiple copies of DNA

Q. 9

Which of the following is/are true about PCR except:

 A

Uses heat labile DNA polymerase

 B

Uses heat stable DNA polymerase

 C

Is technique for DNA amplification

 D

Used to yield multiple copies of DNA

Ans. A

Explanation:

Ans: a. Uses heat labile DNA polymerase,[Ref Harper 30th/458-59; Lippincott 6th/479-83, 5th/497-83; Chatterjea er Shinde7th/267-272]

  • SpecificityQ is based on the use of two oligonucleotide primers that hybridize to complementary sequence on opposite strands of DNA & flank the target sequence Double stranded DNA can be disrupted by heat or high pH, giving rise to single stranded DNA. The single stranded DNA serves as a template for synthesis of a complementary strand by replicating enzymes, DNA polymerase.
  • Early PCR reaction used an E. coli DNA polymerase that was destroyed by each heat denaturation cycle. Substitution of a heat-stable DNA polymerase (Taq polymeraseY from Thermus aquaticus, obviates this problem & has made possible automation of the reaction, since the polymerase reactions can be run at 70°C

Quiz In Between



Hepatorenal Syndrome

HEPATORENAL SYNDROME


HEPATORENAL SYNDROME

  • HRS is development of acute renal failure due to severe hepatic (advanced cirrhosis) or bilary disease with jaundice.
  • Low cardiac putput and high plasma rennin predicts development of HRS.
  • Patient develops oliguria, azotaemia and hyponatraemia.

ETIOLOGY-

  • Bile salt sludging in the tubules
  • Absorption of toxins
  • Increase ADH release
  • Hypoperfusion and renal ischaemia
  • Precipitated by surgery, stress

 PATHOLOGY-

  • Increase in renal vascular resistance along with reduction in systemic vascular resistance
  • Pathogenic marker is intense renal vasoconstriction with vasodilatation

INVESTIGATIONS-  

TYPES-

1. Type 1 HRS-

  • Oliguria
  • Decrease serum creatinine
  • Poor prognosis
  • No proteinuria
  • Urine sodium excretion  <10mmol/day
  • Urine/ plasma osmolarity ration >1.5

Treatment-

  • Albumin + terlipressin

2. Type 2 HRS

  • Refractory ascites
  • Better prognosis
  • Increase serum creatinine levels

Treatment-

  • Terlipressin- DOC
  • Midodrine + pctreotide + IV albumin- reverse renal failure
  • Best therapy for HRS- liver transplantation
  • Dopamine or prostaglandin analogues for renal vasodilation

Exam Important

PATHOLOGY-

  • Increase in renal vascular resistance along with reduction in systemic vascular resistance
  • Pathogenic marker is intense renal vasoconstriction with vasodilatation

INVESTIGATIONS-  

TYPES-

1. Type 1 HRS-

  • Oliguria
  • Decrease serum creatinine
  • Poor prognosis
  • No proteinuria
  • Urine sodium excretion  <10mmol/day
  • Urine/ plasma osmolarity ration >1.5

Treatment-

  • Albumin + terlipressin

2. Type 2 HRS

  • Refractory ascites
  • Better prognosis
  • Increase serum creatinine levels

Treatment-

  • Terlipressin- DOC
  • Midodrine + pctreotide + IV albumin- reverse renal failure
  • Best therapy for HRS- liver transplantation
  • Dopamine or prostaglandin analogues for renal vasodilation
Don’t Forget to Solve all the previous Year Question asked on HEPATORENAL SYNDROME

Module Below Start Quiz

Hepatorenal Syndrome

Hepatorenal syndrome

Q. 1 Albumin treatment along with antibiotic in the setting of SBP(spontaneous bacterial peritonitisis indicated to prevent the development of hepatorenal syndrome is in all , EXCEPT:

 A

Serum creatine is > 1 mg/dl

 B

BUN > 30mg/dl

 C

Total bilirubin is > 4 mg/dl

 D

INR > 2

Q. 1

Albumin treatment along with antibiotic in the setting of SBP(spontaneous bacterial peritonitisis indicated to prevent the development of hepatorenal syndrome is in all , EXCEPT:

 A

Serum creatine is > 1 mg/dl

 B

BUN > 30mg/dl

 C

Total bilirubin is > 4 mg/dl

 D

INR > 2

Ans. D

Explanation:

In patients with SBP along  with cefotaxime albumin infusion is indicated in the setting , when

1.Serum creatine is  > 1 mg/dl
2. BUN >   30 mg/dl
3. Total bilirubin is > 4 mg/dl
 
Dose o f albumin: 1. g/Kg within 6 hours of antibiotic treatment and 1 g/kg  on day 3.
A decrease in mortality from 30%to 10 % is noted.
Ref: AASLD practice  guidelines:Hepatology, Vol.49 ,No.6 ,2009.

Q. 2 Features of Hepatorenal syndrome are

 A

Urine sodium < 10 meq/1

 B

Normal renal histology

 C

Renal function abnormal even after liver become normal

 D

a and b

Q. 2

Features of Hepatorenal syndrome are

 A

Urine sodium < 10 meq/1

 B

Normal renal histology

 C

Renal function abnormal even after liver become normal

 D

a and b

Ans. D

Explanation:

Answer is A & B (urine Na < 10 meq/l and Normal Renal Histology)

Hepatorenal syndrome is associated with normal renal histology and supported by a urine sodium excretion l0meq/L

Hepatorenal syndrome

  • Hepatorenal syndrome is defined as a state of functional renal failure (Reduced GFR) in patients with severe liver disease
  • Structurally /Histologically the kidneys are normal and recover function after successful liver transplantation.
  • The pathogenetic hallmark of hepatorenal syndrome is intense renal vasoconstriction with coexistent systemic vasodilatation
  • The diagnosis of hepatorenal syndrome is considered in accordance with the following diagnostic criteria.

Diagnostic of Hepatorenal Syndrome

Major criteria

  • Low glomerular filtration rate. as indicated by serum creatinine > 1.5 mg/dL or 24-hr creatinine clearance < 40 mL/min
  • Absence of shock, ongoing bacterial infection, fluid losses, and current treatment with nephrotoxic drugs
  • No sustained improvement in renal function (decrease in serum creatinine to 1.5 nig/dL or increase in creatinine clearance to 40 mL/min) after diuretic withdrawal and expansion of plasma volume with 1.5L of a plasma expander
  • Proteinuria mg/d1, and no uhrasonographic evidence of obstructive uropathy or parenchymal renal disease Additional criteria
  • Urine volume < 500 mL/d
  • Urine sodium < 10 meq/L
  • Urine osmolality greater than plasma osmolality
  • Urine red blood cells <50/high- power. field
  • Serum sodium concentration < 130 niEqL

Note: All major criteria must be present for the diagnosis of hepatorenal syndrome.

Additional criteria are not necessary for the diagnosis but provide supportive evidence.


Q. 3 Which of the following statements is incorrect with regard to Hepatorenal syndrome in a patient with cirrhosis

 A

Createnine clearance < 40 ml/min

 B

Urinary sodium < 10mq/L

 C

Urine osmolality lower than plasma osmolality

 D

No sustained improvement in renal function after volume expansion.

Q. 3

Which of the following statements is incorrect with regard to Hepatorenal syndrome in a patient with cirrhosis

 A

Createnine clearance < 40 ml/min

 B

Urinary sodium < 10mq/L

 C

Urine osmolality lower than plasma osmolality

 D

No sustained improvement in renal function after volume expansion.

Ans. C

Explanation:

Answer is C (Urine osmolality is lower than plasma osmolality):

Hepatorenal syndrome is associated with urine osmolality greater than plasma osmolality (and not lower than plasma osmolality).

creatinine clearance < 40 ml/minute and poor response to volume expansion are major diagnostic features of hepatorenal syndrome while urinary sodium of less than 10 mmol/L is an additional criteria that provides supportive evidence.

Quiz In Between


Q. 4

Hepatorenal syndrome is characterized by all of the following except:     
March 2005

 A

Reduction in creatinine clearance

 B

Managed effectively by renal vasodilating agents.

 C

Proteinuria less than 500 mg/ d

 D

Normal intrinsic kidney

Q. 4

Hepatorenal syndrome is characterized by all of the following except:     
March 2005

 A

Reduction in creatinine clearance

 B

Managed effectively by renal vasodilating agents.

 C

Proteinuria less than 500 mg/ d

 D

Normal intrinsic kidney

Ans. B

Explanation:

Ans. B: Managed effectively by renal vasodilating agents.

HRS is defined as worsening azotemia with avid sodium retention and oliguria in the absence of identifiable specific cause of renal dysfucntion in setting of acute or advanced chronic liver disease

No specific tests establish the diagnosis of HRS.

Diagnosis of HRS is based on the presence of a reduced GFR in the absence of other causes of renal failure in patients with chronic liver disease. The following criteria help to diagnose HRS:

Major criteria: All major criteria are required to diagnose HRS.

  • Low GFR, indicated by a serum creatinine level higher than 1.5 mg/ dL or 24-hour creatinine clearance lower than 40 mL/ min
  • Absence of shock, ongoing bacterial infection and fluid losses, and current treatment with nephrotoxic medications
  • No sustained improvement in renal function (decrease in serum creatinine to40 mL/ min) after diuretic withdrawal and expansion of plasma volume with 1.5 L of plasma expander
  • Proteinuria less than 500 mg/ d and no ultrasonographic evidence of obstructive uropathy or intrinsic parenchymal disease

Additional criteria: Additional criteria are not necessary for the diagnosis but provide supportive evidence.

  • Urine volume less than 500 mL/d
  • Urine sodium level less than 10 mEq/ L
  • Urine osmolality greater than plasma osmolality
  • Urine red blood cell count of less than 50 per high-power field
  • Serum sodium concentration less than 130 mEq/ L

The best therapy for HRS is liver transplantation.


Q. 5 All are true about hepatorenal syndrome except:

 A

Creatinine level raised

 B

Albumin infusion given

 C

Liver transplantation improves renal functions

 D

May occur in cirrhosis

Q. 5

All are true about hepatorenal syndrome except:

 A

Creatinine level raised

 B

Albumin infusion given

 C

Liver transplantation improves renal functions

 D

May occur in cirrhosis

Ans. E

Explanation:

Answer- E. Low dose dopamine infusion is very effective

  • The hepatorenal syndrome (HRS) is a form of functional renal failure without renal pathology that occurs in about 10% of patients with advanced cirrhosis or acute liver failure.
  • There are marked disturbances in thc arterial renal circulation in Patients with HRS.
  • TyPe I HRS- a significant reduction in creatinine clearance within 1-2 weeks of presentation.
  • Type 2 HRS- an elevation of serum creatinine level.
  • HRS is often seen in patients with refractory ascites.

Treatment-

  • dopamine or prostaglandin analogues were used as renal vasodilating medications.
  • Patients are treated with midodrine, an alpha-agonist, along with octreotide and intravenous albumin.
  • The best theragy for HRS is liver transplantation.

Quiz In Between



Interstitial Cystitis

INTERSTITIAL CYSTITIS


INTERSTITIAL CYSTITIS (HUNNER’S ULCER, ELUSIVE ULCER)

  • Inflammation of bladder mucosa is cystitis.
  • The characteristic linear bleeding ulcer is caused by splitting of mucosa when the bladder is distended under anaesthesia
  • Severe fibrosis along with inflammation of urinary bladder due to pancystitis results in small thimble bladder.
  • Ulceration of the mucosa occurs in fundus of bladder.
  • Mast cells are often seen in the mucosa, lamina propria, and muscularis propria in interstitial cystitis.
  • Decrease bladder capacity 30- 60 ml

CLINICAL FEATURES-

  • Increase frequency
  • Initial symptom- increase frequency and pain is relieved by micturition and aggrevated by distension of bladder due to decrease bladder capacity
  • Haematuria

INVESTIGATIONS-

  • Cystoscopy and biopsy confirms diagnosis

TREATMENT-

  • Hydrostatic dilatation
  • Instillation of dimethyl sulphoxide
  • Ranitine instillation
  • Ileocystoplasty or caecocystoplasty increases bladder capacity

Exam Important

  • Inflammation of bladder mucosa is cystitis.
  • The characteristic linear bleeding ulcer is caused by splitting of mucosa when the bladder is distended under anaesthesia
  • Severe fibrosis along with inflammation of urinary bladder due to pancystitis results in small thimble bladder.
  • Ulceration of the mucosa occurs in fundus of bladder.
  • Mast cells are often seen in the mucosa, lamina propria, and muscularis propria in interstitial cystitis.
  • Decrease bladder capacity 30- 60 ml
Don’t Forget to Solve all the previous Year Question asked on INTERSTITIAL CYSTITIS

Module Below Start Quiz

Interstitial Cystitis

Interstitial Cystitis

Q. 1 Cells involved in interstitial cystitis?

 A

Lymphocytes

 B

Neutrophils

 C

Macrophages

 D

Mast cells

Q. 1

Cells involved in interstitial cystitis?

 A

Lymphocytes

 B

Neutrophils

 C

Macrophages

 D

Mast cells

Ans. D

Explanation:

Ans. is ‘d’ i.e., Mast cells

  • Mast cells are often seen in the mucosa, lamina propria, and muscularis propria in interstitial cystitis.

Quiz In Between



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