BIOCHEMISTRY & BIOMEDICAL SCIENCES

PBL case objective
a) Discuss an overview of bilirubin metabolism
b) Discuss neonatal physiological jaundice
c) Discuss inborn errors of bilirubin metabolism
d) Discuss biochemical/molecular diagnosis

Case Presentation :
A 48-year-old woman had a history of jaundice for decades, with no related symptoms. A laparoscopic evaluation showed a smooth liver with normal consistency and morphologic features but with a grossly black appearance. A biopsy specimen revealed coarse, deep-brown, pigmented granules on periodic acid–Schiff staining primarily at the canalicular pole of the hepatocytes and especially in the pericentral zones. The laboratory investigation results are given below
Serum Total bilirubin - 4.6 mg/dL
Direct-reacting bilirubin -2.4 mg/dL
Aspartate transaminase - 38 U/L
Alanine transaminase - 25 U/L
Alkaline phosphatase - 45 U/L
Serum Albumin- 4.2 gm/dL
Prothrombin time- 12 seconds

Expression of the multidrug resistance associated protein 2 (MRP2) was absent on anti–MRP2 immunohistochemical analysis

Diagnosis- Dubin–Johnson syndrome was confirmed. This syndrome is an autosomal recessive disorder that is caused by a mutation in MRP2 that results in deficient canalicular expression of MRP2 and impaired secretion of conjugated bilirubin into the bile.

Overview of Bilirubin metabolism

The metabolism of bilirubin can be divided into three different steps:

i) Uptake: The bilirubin is formed in reticuloendothelial cells and released into the circulation. In circulation, the bilirubin binds to albumin at high and low-affinity binding sites and transported to the liver cells. In the hepatocytes, the bilirubin is rapidly sequestered by protein Ligandin Y which enables uptake of bilirubin into hepatocytes against the concentration gradient.

ii) Conjugation: In hepatocytes, the bilirubin is conjugated to UDP-glucuronic acid form bilirubin mono-glucuronides and subsequently converted to bilirubin diglucuronides. The reaction is catalyzed by an inducible enzyme UDP-glucuronosyl transferase (UGT).

iii) Secretion: The conjugated bilirubin is secreted into canalicular space through multidrug resistance transporter protein. The secreted conjugated bilirubin enters the gallbladder and secreted via bile into the intestine.

Figure 1: Overview of bilirubin metabolism

Inherited disorders of bilirubin metabolism:

Gilbert Syndrome:

The Gilbert syndrome is characterized by unconjugated hyperbilirubinemia with serum bilirubin less than 3.0 mg/dL. Gilbert syndrome is associated with a defect in bilirubin uptake and the genetic mechanism is not fully understood. Studies have shown that the mutation in the promoter region of UTGA1A and other missense mutation is associated with Gilbert syndrome. This leads to decreased activity of UGT enzymes in the liver resulting in unconjugated hyperbilirubinemia. This genetic disorder remains undiagnosed until adulthood because of milder hyperbilirubinemia and no specific jaundice. In patients with Gilbert syndrome, fasting causes an increase in serum bilirubin, coloration of eyes and sclera. This is mainly due to the competitive displacement of bilirubin bound to albumin by fatty acid. The liver enzymes are within normal reference interval.

Crigler Najjar Syndrome I:

The Crigler Najjar syndrome-I is characterized by a complete deficiency of enzyme UGT leading to unconjugated hyperbilirubinemia with serum bilirubin above 20 mg/dL. The unconjugated bilirubin above 20 mg/dL crosses the blood-brain barrier; and causes kernicterus. The diagnosis is based on bilirubin greater than 20 mg/dL and normal liver enzymes including aspartate transaminase, alanine transaminase, alkaline phosphatase. Phototherapy is used for reducing bilirubin as it makes bilirubin more soluble in water.

Crigler Najjar Syndrome II:

The Crigler Najjar Syndrome II is characterized by a partial deficiency of enzyme UGT resulting in increased unconjugated bilirubin (5-20 mg/dL). In addition, the unconjugated bilirubin is lowered upon administration of phenobarbital by inducing UGT enzyme. The patients with Type I deficiency are non-responsive to phenobarbital. Apart from unconjugated hyperbilirubinemia, all liver enzyme parameters are normal and kernicterus is absent.

Dubin Johnson syndrome:

Dubin Johnson syndrome is characterized by conjugated hyperbilirubinemia due to defective MRP2 protein. MRP2 protein is responsible for secretion of bilirubin to bile canaliculi. The normal liver enzymes including aspartate transaminase, alanine transaminase, alkaline phosphatase suggestive of normal liver function without any cholestasis. The pigmentation of the liver is observed.

Rotor syndrome:

Rotor syndrome is characterized by conjugated hyperbilirubinemia due to defective secretion of bilirubin to bile canaliculi but the etiology is not known. The normal liver enzymes including aspartate transaminase, alanine transaminase, alkaline phosphatase suggestive of normal liver function without any cholestasis. No pigmentation of the liver is observed.

Take Away Points

Bilirubin conjugation occurs in the liver and the reaction is catalyzed by an inducible enzyme UGT.
Conjugation and phototherapy make bilirubin more water soluble.
Measurement of unconjugated and conjugated bilirubin is used for diagnosis of inherited hyperbilirubinemia.

Reference:
Moni et al. 2016 NEMJ

PBL case objective
a)Differential Diagnosis of Jaundice
b)Biochemical basis for diagnosis of acute liver disease
c) Biochemical basis for diagnosis of obstructive jaundice

Clinical case 1: A 52-year-old man presented with a history of nausea, vomiting, and abdominal pain and icteric jaundice for one week. A physical examination revealed stable vital signs, and there was no evidence of hepatic encephalopathy. He had icteric sclera and right upper quadrant abdominal tenderness to palpation.The laboratory investigation results are presented below.
Total serum Bilirubin - 15.4 mg/dL
Direct bilirubun-11.5 mg/dl
Aspartate transaminase- 720 U/L
Alanine transaminase - 1625 U/L
Alkaline Phosphatase- 211 U/L
Total Protein- 7.3 g/dl
Serum Albumin- 3.8 g/dl
Prothrombin time : 12 seconds

Clinical Case 2. A 34-year-old woman presented with a history of jaundice, fever, and abdominal pain. Physical examination showed scleral icterus and right upper quadrant tenderness without inspiratory arrest at palpation. The laboratory investigation results are presented below.
Total Bilirubin: 5.2 mg/dL
Direct Bilirubin: 4.5 mg/dL
Alanine aminotransferase: 180 U/L
Aspartate aminotransferase:210 U/L
Alkaline phosphatase: 750 U/L,
gamma-glutamyltransferase: 999 U/L

Discussion:

Differential Diagnosis of Jaundice

The jaundice is defined as the discoloration of skin, eye, and sclera with serum bilirubin >3mg/dL. Based on the etiology, jaundice can be classified into pre-hepatic (hemolytic jaundice), hepatocellular jaundice and post-hepatic (obstructive) jaundice. In pre-hepatic jaundice, the unconjugated bilirubin markedly increased in the serum because the production of bilirubin exceeds the conjugation capacity of the liver. In hepatocellular jaundice, the unconjugated and conjugated bilirubin is increased because of leakage of bilirubin from affected hepatocytes. The increase in bilirubin is accompanied by markedly increased alanine transaminase and aspartate transaminase. Measurement of albumin and prothrombin time helps to differentiate chronic liver disease from an acute condition. In case of obstructive jaundice, the conjugated bilirubin is increased due to obstructed biliary flow and regurgitation of bilirubin. An elevated serum alkaline phosphatase and 5'-nucleotidase activity are suggestive of obstructive jaundice.

Differential Diagnosis of Jaundice (Adapted from Harrison's Internal Medicine-16th ed)

Biochemical basis for diagnosis of acute liver disease
Case 1: The clinical case presentation and biochemical test results are suggestive of acute hepatitis. The total and direct bilirubin are markedly increased. The transaminases are also elevated with ALT >20 times upper reference limit shows that the viral infection may be caused lysis of hepatocytes and leakage of bilirubin and transaminases.  In addition, serum albumin and prothrombin time are within normal reference interval suggesting the acute nature of the disease. To confirm the etiology and type of infected virus, the antibody detection assay against different class hepatitis virus, serological test and PCR identification of virus serotype are recommended.

In chronic hepatic diseases, the ALT and AST are elevated (generally less than 7X URL), a decrease in serum albumin and prolonged prothrombin time. The decreased serum albumin and prolonged prothrombin time are a result of a compromised liver function.

Biochemical basis for diagnosis of Obstructive jaundice
Case 2: The clinical case presentation and biochemical test results are suggestive of obstructive jaundice. The total and direct bilirubin mildly increased with significant hyperbilirubinemia. The transaminases  (ALT & AST) are mildly elevated with approximately 4-5 X URL with the marked rise in alkaline phosphatase and gamma-glutamyl transferase. The direct hyperbilirubinemia with elevates serum alkaline phosphatase and gamma-glutamyl transfer are suggestive of cholelithiasis.  The enzyme activity of ALP is also elevated in Paget disease (bone) and cancer etc. GGT is also not the specific marker for obstructive jaundice and elevated in patients with the alcoholic liver disease. Therefore, measurement of 5'-nucleotidase and ultrasonography of liver and gallbladder area are preferable for confirmatory diagnosis.

Summary

The measurement of bilirubin, liver enzymes and enzymes of biliary track enable the differential diagnosis of hepatocellular and obstructive jaundice. The measurement of albumin and prothrombin time provide additional evidence on occurrence and duration of disease. The detection and identification of viral serotypes in acute viral hepatitis are important for treatment of viral hepatitis. Advancement in medicine has made the treatment of some type of hepatitis possible.

PBL Case Objective
a) Describe how alcohol is metabolized in the body.
b) Discuss the cause of hematological abnormalities if any.
c) Discuss how biochemical results correlate with the provisional diagnosis.
d) Discuss any test recommendations.
e) Discuss the provisional diagnosis.

Clinical Presentation
A 40 years old man was admitted to the hospital with hematemesis, loss of consciousness, swelling of lower limbs. The patient had a history of alcoholism with daily consumption of approximately 1 to 2 L of beer every day for the past ten years. the physical examination showed hepatomegaly, mild ascites. The hematological and biochemical results are presented below.










Discussions
Alcohol Metabolism occurs in the liver
The liver is the major tissue for alcohol metabolism in the body. Before alcohol reaches the liver, alcohol dehydrogenase isoform present in the stomach metabolizes minor quantity of alcohol. The quantity of alcohol metabolized in the stomach depends on the fed state, gastric emptying time. During fasting, the alcohol rapidly reaches the small intestine and absorbed into the bloodstream which increases the bioavailability.

In the liver, two enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) catalyzes the conversion of ethanol to acetyl CoA and NAD+ are reduced to generate NADH. The genetic polymorphism of ADH and ALDH genes have associated the activity of the ADH and ALDH enzymes and subsequently sensitivity to alcohol. The studies have shown ethnic variations in alcoholism and alcoholism associated disorders. The individuals with increased ADH activity or decreased ALDH activity may have increased accumulation of aldehyde and a higher risk of liver diseases.

The second enzyme CYP2E1 metabolizes ethanol when blood concentration of alcohol is higher. The CYP2E1 (microsomal ethanol oxidizing enzyme )is a cytochrome P450 family enzyme that has a low affinity (Ka= 10nM) for ethanol. This inducible enzyme is more active in an individual with low ADH activity or chronic alcohol consumption. The other minor pathways for alcohol metabolism have been reported but the physiological significance is unknown.



Figure 1: Metabolism of Ethanol. Alcohol is metabolized in the liver to form acetate. The formation of acetaldehyde is catalyzed by two separate enzymes depending on the ethanol concentration in the body. Alcohol dehydrogenase an enzyme catalyzes the formation of acetaldehyde with the generation of NADH. The second enzyme CYP2E1 catalyzes the same reaction but requires NADPH for its activity.

Metabolic changes and Liver Pathology:

The oxidation of alcohol in the liver generates the acetyl CoA& NADH and decreases the ratio of CoA/ acetyl CoA & NAD+/NADH. The altered equilibrium of these metabolites result in metabolic adaptations of cells and chronic exposure lead to liver diseases. The metabolic adaptations in the liver include:
i) Alcohol metabolism alters the hepatic redox state
Alcohol dehydrogenase and Aldehyde dehydrogenase generates each generates one molecule of NADH in the process of converting ethanol to acetate. The decreased NAD+/NADH ratio resulting from increased cellular NADH disrupts redox equilibrium and dysregulation of various glucose and lipid metabolic pathway. The availability of NADH and acetyl CoA inhibits the energy-liberating pathways such as glycolysis, tricarboxylic acid cycle, fatty acid oxidation, pyruvate oxidation.
On the other hand, cells attempt to normalize the NAD+/NADH ratio by favoring the conversion of pyruvate to lactate and ketogenic pathway. The conversion of pyruvate to lactate partially rescues the NAD+/NADH but the chronic exposure decreases the availability pyruvate for a gluconeogenic pathway, hypoglycemia, and lactic acidosis.
ii) Alcohol metabolism increases triacylglycerol synthesis, lipid storage, and ketogenesis
In the liver, alcohol increases fatty acid by inhibiting beta-oxidation of fatty acid. The increased concentration of glyceraldehyde-3-phosphate and free fatty acid enhance the formation of triacylglycerol and secretion of VLDL. In severe alcoholism, the excess acetyl CoA generated is utilized for ketogenesis and released into the bloodstream resulting in ketoacidosis.

iii) Excessive chronic alcohol consumption can cause fatty liver, hepatocellular damage, cirrhosis
The hepatocellular damage can affect the liver function by decreasing liver function such as synthesis of proteins and clotting factors, detoxification of bilirubin and ammonia etc. The hepatomegaly, gross histological arrangement of hepatocytes and altered serum bilirubin and enzymes are the characteristic feature of liver cirrhosis.


Figure 2: Metabolic derangement in alcoholic liver disease

Disease correlation with Clinical Chemistry Parameters
The clinical chemistry parameters can be categorized into three different categories:
i) Assessing synthetic function
The synthetic function of the liver may be assessed by measuring the albumin and clotting factor. The albumin constitutes 60% of the plasma and exclusively synthesized and secreted by the liver. In addition, albumin has a half-life of approximately 21 days that allows monitoring of chronic defect in the liver. The caveat is that albumin may be lowered in other conditions such as malnutrition, proteinuria. Therefore, the measurement of albumin with prothrombin time can provide a better understanding of defective tissue. In this particular clinical case, the albumin is lower with prolonged prothrombin time suggesting a chronic hepatocellular disease. The acute hepatocellular disease differs from a chronic form with normal albumin and prothrombin time.

ii) Assessing detoxification function
Bilirubin is conjugated by enzyme UDP-glucuronosyl transferase and excreted via bile. The majority of ammonia are handled by the liver and converted into urea in the liver. Therefore, the evaluation of bilirubin, urea, and ammonia are used to assess the detoxification function of the liver. Elevated urea and ammonia are suggestive of defective excretion these metabolites whereas increased ammonia and bilirubin with normal or blood urea are suggestive of the hepatocellular disease.

c) Assessing hepatocellular damage
The extent of hepatocellular damage can be assessed using liver enzymes ALT and AST. Although both ALT and AST are not a specific marker for liver injury and elevated during muscle injury, interpretation of AST/ALT ratio (de Ritis ratio) along with ALP and GGT provides liver injury. During acute viral hepatitis, ALT is rapidly elevated and the activity in serum is higher than AST. In contrast, the AST is mildly elevated with higher serum activity compared to ALT (AST/ALT ratio >2) is suggestive of the chronic alcoholic liver disease. In addition, mild elevated of GGT also suggest the chronic alcoholic liver disease.

d) Decreased hemoglobin concentration:
The hemoglobin is synthesized in bone marrow in response to erythropoietin. During chronic liver disease, the erythropoietin synthesis is also decreased leading to decreased hemoglobin.

Additional Investigation recommended for treatment prognosis
CAGE questionnaire for alcohol dependency
Assessment of proteinuria and serum creatinine (renal function)
Histopathological examination of liver
Carbohydrate-deficient transferrin

Provisional Diagnosis
The case presentation and laboratory results suggestive of chronic alcoholism with hepatocellular damage.

Reference
AASLD practice guidelines Alcoholic liver disease
Cederbaum AI 2002, Clinical liver disease
McPherson et al, 2006 BMJ

1) Plasma proteins are composed of mostly glycoproteins, lipoproteins, and immunoglobulins that function in the immune system; binding and transport of nutrients, toxic substances, metals etc. The normal reference range for total plasma proteins are

a) 2.5-4.5 gm/dl
b) 4.5-6.0 gm/dl
c) 6.0-8.3 gm/dl
4) >8.3gm/dl

2) Plasma proteins are broadly classified as albumin and globulin (A&G) and reference range for A: G ratio is 0.8-2.0. Based on the electrophoretic mobility, the globulin fraction may be further classified into alpha-1 globulin, alpha-2 globulin, beta-globulin, and gamma-globulin. Which of the following protein is present in the gamma-globulin fraction?

a) Transthyretin
b) Ceruloplasmin
c) Haptoglobin
d) Immunoglobulin

3) C-reactive protein, a plasma protein that is elevated during inflammation and infections. C-reactive protein falls into the category

a) Transport proteins
b) Clotting proteins
c) Plasma Enzymes
d) Acute phase proteins

4) Albumin (69kDa) is the major plasma protein constituting 60% of total plasma proteins. Which of the following is not the function of albumin

a) Maintenance of osmotic pressure
b) Binding and transport of fatty acids and bilirubin
c) Transport of iron
d) Transport of drugs such as sulphonamides

5) Which of the following protein has a half-life of approximately 48 hours and also measured as a biomarker for acute hepatic failure or malnutrition.

a) Albumin
b) Transthyretin
3) Ceruloplasmin
d) Haptoglobin

6) Which of the following plasma protein is not involved in iron homeostasis?

a) Haptoglobin
b) Transferrin
c) Ferritin
d) Ceruloplasmin

7) Clotting factors are plasma proteins responsible for blood coagulation and deficiency results in bleeding disorder and prolonged prothrombin time (PT). The prolong PT can also occur due to deficiency of vitamin K. The hepatic vs extrahepatic etiology for prolonged PT can be determined by

a) measuring of factor VIII activity
b) measuring International Normalized Ratio (INR)
c) parenteral injection of 10 mg Vit K
d) measuring factor IX activity

8) Primary hemochromatosis is caused by a mutation of gene HFE and characterized by iron overload in various tissues. Which of the following condition does not co-exist with primary hemochromatosis?

a) Bronze diabetes
b) Hepatic cirrhosis
c) Cardiac abnormalities
d) Anemia

9) The normal reference range for prothrombin time is > 15 second. The following condition have PT above normal reference range

a) Wilson disease
b) Ischemic hepatic disease
c) Primary Hemochromatosis
d) Autoimmune hepatitis

10) The following condition has altered A: G (albumin/globulin) ratio less than 2 with decreased plasma albumin concentration:

a) Chronic hepatic Cirrhosis
b) Acute hepatitis
c) Obstructive jaundice
d) Increased alcohol consumption

Answers
1-c) 6.0-8.3 gm/dl
2- b) Ischemic hepatic disease
3-d) Acute phase proteins
4-c) Transport of iron
5-b) Transthyretin
6-d) Ceruloplasmin
7- c) parenteral injection of 10 mg Vit K
8-d) Anemia
9-b) Ischemic hepatic disease
10-a) Chronic hepatic Cirrhosis

Multiple Choice Question  on 

Bilirubin Metabolism and Related Disorders

1) Bilirubin is produced from catabolism of protoporphyrin IX by a microsomal enzyme in a two reaction step. The first step involves the opening of the tetrapyrrole ring to form biliverdin which is further reduced to form bilirubin. What are the enzymes responsible for these two steps?

a) Heme dehydrogenase and Bilirubin oxygenase
b) Heme oxygenase and Biliverdin reductase
c) Heme deoxygenase and Biliverdin reductase
d) Heme oxygenase and Bilirubin reductase

2) Approximately 85% of the total bilirubin produced are derived from senescent red blood cells in the reticuloendothelial cells of liver, spleen and bone marrow. The remaining 15% of bilirubin is produced from RBC precursor during ineffective erythropoiesis, myoglobin, cytochromes, and peroxidases. The daily production of bilirubin is

a) 500-1000 mg
b) 100-250 mg
c) 250-350 mg
d) 0-100 mg

3) The bilirubin produced in spleen and bone marrow diffuses into the blood, binds to albumin transported to the liver and binds to Ligandin Y protein (a GST gene family protein) inside the hepatocytes. The bilirubin is transported into the hepatocytes via

a) passive diffusion of bilirubin into hepatocytes
b) carrier-mediated saturable system
c) active transport coupled with Na K ATPase
d) MRP2 protein

4) The unconjugated bilirubin (non-polar form) is converted into more polar form by conjugating with glucuronic acid. The stepwise addition of two glucuronic acids to bilirubin result in bilirubin diglucuronides. The enzyme that catalyzes the formation of bilirubin diglucuronide is

a) Bilirubin UDP-glucuronides synthase
b) Bilirubin UDP-glucuronides mutase
c) Bilirubin UDP-glucuronosyltransferase
d) Bilirubin UDP-glucuronosyl isomerase

5) The secretion of bilirubin from hepatocytes to canaliculi is an energy-dependent process. The transporter protein involved in this protein is

a) MRP2 protein
b) active transport coupled with Na K ATPase
c) Bilirubin transporting protein
d) Chylomicron

6) In the intestine, bacterial degradation of bilirubin forms urobilinogen. Urobilinogen is a colorless bilirubin derived product which is further oxidized to form the following pigments except

a) Urobilin
b) Mesobilin
c) Stercobilin
d) Exobilin

7) Gilbert's syndrome is the genetic disorder of bilirubin metabolism and the defect lies in the uptake of bilirubin. Which of the following form of bilirubin is elevated in these subjects?

a) Direct bilirubin and Total bilirubin
b) Indirect and total bilirubin
c) Both direct and indirect bilirubin
d) None

8) Crigler Najjar Syndrome is the inherited metabolic disorder of bilirubin metabolism due to a defective enzyme

a) Heme oxygenase
b) Biliverdin reductase
c) UDP-Glucuronosyl transferase
d) beta-glucuronidase

9) Urobilinogen is oxidized to form a colored product and these give the characteristic color of urine and feces. In which type of jaundice, the stool color is clay-colored because of lack of urobilin?

a) Hemolytic jaundice
b) Viral hepatitis
c) Obstructive jaundice
d) Alcoholic cirrhosis

1) Most of the plasma proteins are synthesized in the liver. Which of the following plasma protein is not synthesized in the liver?

a) Albumin
b) Transferrin
c) Ceruloplasmin
d) von Willebrand factor

2) Which of the following is not the metabolic function of the liver?

a) Synthesis and assembly of chylomicrons
b) Synthesis and secretion of bile acids
c) Synthesis and breakdown of glycogen
d) Gluconeogenesis

3) Bile acids are conjugated to amino acid or taurine to form bile salts. Which of the following amino acid is utilized for conjugation reaction?

a) Alanine
b) Glycine
c)Valine
d)Leucine

4) Cholic acid and Chenodeoxycholic acid are the primary bile acid synthesized in the liver. The bile acid is converted into secondary bile acids (eg. deoxycholic acid) by

a) Enzymes present in the gall bladders
b) Enzymes present in the duodenum
c) Enzymes produced by exocrine pancreas
d) Bacterial oxidation in the small intestine

5) Which of the following are functions of bile salts except

a) Formation of bile salts decrease the passive absorption in the small intestine
b) Enables active transport of bile salts in the terminal ileum
c) Allows solubilization and elimination of cholesterol
d) Increases emulsification of carbohydrates in the small intestine

6) The bile salt synthesis is subjected to feedback regulation by bile salt reabsorbed from ileum and recirculated to the liver. Which of the following enzyme is the rate-limiting enzyme of bile acid synthesis?

a) 7-alpha-hydroxylase
2) 12-alpha-hydroxylase
3) 12-beta-hydroxylase
4) 5-beta isomerase

7) Bile juice secreted by the gallbladder is composed of bile salts, bilirubin, cholesterols. Which
of the following is not true regarding hepatic bile formation?

a) Primary bile salts are predominantly present compared to secondary bile salts.
b) Glycine conjugates predominate in a ratio of approximately 3:1 to 4:1.
c) Free bile acids are normally present in the bile.
d) Bile acids are transported across the canicular surface via carrier-mediated active transport

8) The regulation of bile salt metabolism is mediated via

a) Feed-back inhibition of enzyme activity of 7-alpha-hydroxylase
b) Feed-back inhibition of enzyme activity of 12-alpha-hydroxylase
c) Bile salt binding to farnesoid X receptor and suppression of 7-alpha-hydroxylase gene expression
d) Bile salt binding to farnesoid X receptor and suppression of 12-alpha-hydroxylase gene expression

Multiple Choice Question on Metabolism and Disposal of  Urea, Creatinine and Uric Acid

1) The urea molecule consists of two nitrogens and one carbon atom. The first nitrogen atom is derived from the free ammonium ion and second from an amino acid. The amino acid that donates the  second amino group for the formation of urea is

a) Arginine
b) Aspartate
c) Glutamate
d) Ornithine

2) The free ammonium group  (NH4+) is derived from the following processes:

a) Oxidative deamination of Glutamine catalyzed by enzyme glutamate dehydrogenase
b) Deamidation of histidine catalyzed by the enzyme histidase
c) Deamidation of serine and threonine by a PLP requiring enzyme serine dehydratase
d) All of the above

3) The first step in the urea cycle is a condensation of CO2, ammonia, and ATP to form carbamoyl phosphate. The following are true regarding the formation of carbamoyl phosphate except:

a) The reaction is catalyzed by a rate-limiting enzyme carbamoyl phosphate synthase-I
b) The presence N-acetyl glutamate inhibits the activity of CPS I enzyme
c) Two molecules of ATP are required for this reaction
d) The reaction occurs in mitochondria

4) Which of the following amino acid: keto acid pair bridge the urea cycle with the tricarboxylic acid cycle for the maintenance of nitrogen and amino acid pool

a) Glutamate: Alpha-ketoglutarate
b) Aspartate: Oxaloacetate
c) Alanine: Pyruvate
d) None of the above

5) Four Adenosine triphosphate equivalents are required for a formation of urea in the urea cycle. Which of the following enzymes utilize the ATPs

a) Carbamoyl Phosphate synthase I
b) Ornithine Transcarbamoylase
c) Argininosuccinate synthase
d) Ariginosuccinase
a) Carbamoyl Phosphate synthase I
b) Ornithine Transcarbamoylase
c) Argininosuccinate synthase
d) Ariginosuccinase

8) The deficiency of which of the following enzyme causes Citrullinemia- a condition with an accumulation of citrulline in the blood

a) Carbamoyl Phosphate I
b) Ornithine Transcarbamoylase
c) Argininosuccinate synthase
d) Ariginosuccinase

9) Which of the following compound is administered to the patients with urea cycle defect to remove the ammonia?

a) Inulin
b) Phenylbutyrate
c) Arginine
d) Ornithine


10) The following conditions lead to the hyperammonemia
a) Chronic Liver Cirrhosis
b) GI bleeding
c) Deficiency of CPS I
d) All of the above

11) The creatine synthesis begins in the kidney and is completed liver and require three different amino acid for its formation.  The creatine undergoes phosphorylation to form creatine phosphate in various tissue. The following amino acids are required for its synthesis EXCEPT:

a) Alanine
b) Arginine
c) Glycine
d) Methionine

12) The interconversion of creatine and creatine phosphate is catalyzed by an enzyme creatine kinase. The creatine kinase has three different isoenzymes.  Which of the following isoenzyme is present in heart tissue that is elevated in myocardial infarction?

a) CK-MM
b) CK-MB
c) CK-BB
d) None of the above

13) The interconversion of creatine and creatine phosphate is catalyzed by an enzyme creatine kinase. The creatine kinase has three different isoenzymes.  Which of the following isoenzyme is present in heart tissue that is elevated in skeletal muscle atrophy?

a) CK-MM
b) CK-MB
c) CK-BB
d) None of the above

14) Creatinine is a compound formed by a spontaneous cyclization of creatine phosphate in brain and muscles. The following statement is true for creatinine EXCEPT:

a) One to two percent of Creatine phosphate is irreversibly converted to creatinine
b) The concentration of creatinine is fairly constant and relative to body muscle mass
c) Creatinine is freely filtered through the glomerular membrane
d) Creatinine is not secreted by kidney tubules

15) Creatinine clearance is used for

a) estimation of renal blood flow
b) estimation of glomerular filtration rate
c) evaluation of nephrolithiasis
d) None of the above

16) Uric acid is the excretory end product of purine metabolism. Which of the following condition lead to the increased production of uric acid

a) Hypoxanthine- Guanosine phosphoribosyltransferase
b) Increased lactate levels and chronic alcoholism
c) Acute and Chronic Renal Disease
d) All of the above

d) Allantoin

Multiple Choice Answers
1-b) Aspartate
2-d) All of the above
3- b) The presence N-acetyl glutamate inhibits the activity of CPS I enzyme
4- b) Aspartate: Oxaloacetate
5-a) Carbamoyl Phosphate synthase I &c) Argininosuccinate synthase
6- d) antiport that exchange citrulline with ornithine
7- b) Ornithine Transcarbamoylase
8- c) Argininosuccinate synthase
9- b) Phenylbutyrate
10- d) All of the above
11- a) Alanine
12- b) CK-MB
13- a) CK-MM
14- d) Creatinine is not secreted by kidney tubules
16- d) All of the above
17-c) Glutamine
18-a) Amino acids
19-d) Arginine succinate
b) Uric acid

Multiple Choice Question Set I
1) Caffeine increases the cellular cyclic Adenosine monophosphate (cAMP) that modulate lipolysis.
a) They increase the formation of cAMP from ATP in the cell membrane
b) They promote the transport of cAMP from the endoplasmic reticulum to cytosol
c) They enhance the formation of cAMP from AMP in the cell
d) They inhibit the degradation of cAMP produced in the cell

2) Obesity and Type 2 diabetes are associated with increased hypertriacylglycerolemia caused by insulin resistance. Which of the following is true for this condition?
a) Insulin resistance leads to increased absorption of triacylglycerol
b) Insulin resistance leads to decreased uptake of triacylglycerol in adipose tissues
c) Insulin resistance leads to decreased lipolysis in adipose tissues
d) Insulin resistance leads to decreased uptake to triacylglycerol in hepatocytes.

3) Vitamin A deficiency causes :
a) Loss of fertility
b) Hyperlipidemia
c) DNA damage
d) Xerophthalmia

4) Vit D3 has a direct role in increasing serum calcium level via one of the following organs
a) Intestine
b) Kidney
c) Liver
d) Muscle

5) Parathyroid hormone and Vit D3
a) Increases calcium in serum
b) Increases bone resorption
c) Decreases phosphate excretion
d) Decreasing calcium absorption from the intestine

Multiple Choice Question Set I

1) Which of the following enzyme is a sensitive marker of alcoholic liver disease?
a) Alanine transaminase
b) Aspartate transaminase
c) Alkaline phosphatase
d) Gamma-Glutamyltransferase

2) Which of the following marker is used for the differential diagnosis of obstructive jaundice?
a) Lactate dehydrogenase
b) Creatine Kinase
c) Carbonic anhydrase
d) 5’- Nucleotidase

3) Criggler’s Najjar Syndrome II occurs due to
a) Excessive production of bilirubin
b) The absence of conjugating enzyme
c) Partial deficiency of the conjugating enzyme
d) Overexpression of conjugating enzymes

4) Gluconeogenesis occurs in the liver because of the presence of
a) Phosphofructokinase
b) Pyruvate carboxylase
c) Glucose 6 phosphatase
d) Glucokinase

5) Which the following statements is true of warfarin?
a) competitive antagonist of vitamin
b) co-administration of aspirin is safe
c) prolonged prothrombin time
d) reduced synthesis of coagulation factor VIII.

6) How many amino acids are present in gastrin?
a) 17
b) 27
c) 51
d) 68

7) Starch contains glucose units with 1-4 and 1-6 bonds and which enzyme helps to a hydrolyzed branched chain?
a) amylase
b) maltase
c) isomaltase
d) sucrase

8) Which of the following is an intracellular buffer in our body?
a) Bicarbonate buffer system
b) Albumin buffer system
c) Ammonia buffer system
d) Phosphate buffer system

9)Which of the following molecule is the substrate for ketogenesis?
a) alanine
b) oxaloacetate
c) triacylglycerol
d) acetyl CoA

10) Inhibin participate to play a major role in the production of sperm and testosterone, identify the active inhibin from the following
a) inhibin A
b) inhibin B
c) alpha-inhibin
d) beta - inhibin

Multiple Choice Questions Set I Answers
1-d, 2- d, 3-c, 4-c, 5- a, 6- b, 7-c, 8-d, 9-d, 10-b

Multiple Choice Question Set II

11) Which of the following occur as a result of the secretion of parathyroid hormone in the bloodstream?
a) a decrease in plasma glucose levels
b) an increase in plasma calcium levels
c) an increase in the body’s basal metabolic rate
d) increase in heart rate and blood pressure

12) Which of the following condition is not related to hypocalcemia?
a) Vitamin D deficiency
b) Muscle weakness
c) Cardiac arrhythmia
d) Tetany

13) Which ion is directly related to calcium homeostasis?
a) Mg2+
b) Sodium
c) Phosphate
d) Chloride

14)The class of lipoproteins having a beneficial effect in atherosclerosis is …
a) Low density of lipoproteins
b) Very low density lipoproteins
c) High density lipoproteins
d) Chylomicrons

15) The highest phospholipids content is found in …
a) Chylomicrons
b) VLDL
c) LDL
d) HDL

16) Triglycerides are transported from the liver to extrahepatic tissues by …
a) Chylomicrons
b) VLDL
c) HDL
d) LDL

17) Nascent HDL of intestinal origin acquires Apo C and Apo E from …
a) Chylomicrons
b) VLDL
c) LDL
d) HDL of the hepatic origin

19) Abetalipoproteinaemia occurs due to a block in the synthesis of …
a) Apoprotein A
b) Apoprotein B
c) Apoprotein C
d) Cholesterol

20) Genetic deficiency of lipoprotein lipase causes hyperlipoproteinemia of the following type:
a) Type I
b) Type IIa
c) Type IIb
d) Type V

Multiple Choice Question Set II Answers
11-b, 12-c, 13-c, 14-c, 15-d, 16-b, 17-b, 19-b, 20-b

Multiple Choice Question Set III

21) Which of the following best describes the metabolic acid-base disturbance?
a) metabolic acidosis with a high anion gap
b) metabolic acidosis with a normal anion gap
c) respiratory acidosis with a high anion gap
d) respiratory acidosis with a normal anion gap

22) The short strand of ........ primer is required for the replication of DNA:.
a) DNA
b) RNA
c) Histone
d) hnRNA

23) The following are the post-translational modifications that may be required for trafficking or function of the proteins, except:
a) Hydroxylation
b) Glycosylation
c) Oxygenation
d) Phosphorylation

24) The blotting technique which is used to detect the RNA in a sample is....
a) Southern blotting
b) Eastern blotting
c) Northern blotting
d) Western blotting

25) The hormone which is responsible for the maintenance of the menstrual cycle in women is....
a) Progesterone
b) Prolactin
c) Estrogen
d) Cortisol

26) Among them, which is “palindrome” Sequence?
a) 5’-GATTAC-3’
b) 5’-GCATTA-3’
c) 5’-GTCTAA-3’
d) 5’-GAATTC-3’

27)The base that never found in the genetic code is.....
a) Adenine
b) Guanine
c) Thiamine
d) Cytosine

28) Which of the following is the end product of purines metabolism in humans?
a) Xanthine
b) Uric acid
c) Urea
d) Allantoin

29) What is progesterone supposed to balance out?
a) Testosterone
b) Insulin
c) Estrogen
d) Glucagon

30) The best biochemical indicator of vitamin D status is:
a) serum calcium
b) serum 25‐hydroxyvitamin D3
c) serum 1,25‐dihydroxyvitamin D3
d) serum alkaline phosphatase

Multiple Choice Questions Set III Answer
21-a, 22-b, 23-c, 24-c, 25-a, 26-d, 27-c, 28-d, 29-c, 30-c

Multiple Choice Question Set IV
31) Fatty liver is caused by the accumulation/deposition of fats in the liver. Which of the following is not the likely cause of fatty liver?
a) Obesity
b) Starvation
c) Pregnancy
d) Diabetes mellitus

32) Which of the following is the nonfunctional plasma enzymes increased in alcoholic subjects?
a) Alkaline phosphatase
b) Acid Phosphatase
c) Lactate dehydrogenase
d) Gamma-glutamyl transferase

33) Criggler Najjar syndrome is a genetic disorder associated with unconjugated hyperbilirubinemia. What is the deficient enzyme responsible for the disease?
a) Heme oxygenase
b) Biliverdin reductase
c) Bilirubin UDP-glucuronosyl synthase
d) Bilirubin UDP-glucuronosyl transferase


34) All of the following statement is true concerning urobilinogen except:
a) Produced by oxidative action of intestinal bacteria.
b) Undergoes significant enterohepatic circulation.
c) Urinary levels increased in biliary obstruction.
d) Fecal levels decreased in biliary obstruction.

35) Galactosemia is caused by a deficiency of the enzyme responsible for the galactose metabolism. Which of the following enzyme is deficient?
a) Galactokinase
b) Galactose 1- phosphate uridyl transferase
c) UDP-4- Epimerase
d) Galactose dehydrogenase

36) The Kidney contributes to acid-base balance by:
a) Secretion of ammonia
b) Reclamation of bicarbonate
c) Increased ketogenesis
d) Decreased CO2 uptake

37) Transport of sodium in the renal tubules involves:
a) Na+/K+-ATPase located on the luminal membrane of proximal tubules
b) Na+/K+-ATPase located on the membrane of distal tubules
c) Entry of sodium into tubular cells by passive transport
d) cAMP

38) Which of the following amino acids has an important role in the transport of amino group from peripheral tissues to the liver?
a) Serine
b) Methionine
c) Glutamine
d) Arginine

39) The Renal Plasma flow is best measured by….
a) Inulin Clearance Test
b) GFR estimation
c) PAH Test
d) Creatinine Clearance

40) Which one of the following statement concerning a one-week-old male infant with undetected classic phenylketonuria is correct?
a) Tyrosine is a non-essential amino acid for the infant
b) High levels of phenylpyruvate appear in his urine
c) A diet devoid of phenylalanine should be initiated immediately
d) Therapy must begin within the first year of life.

Multiple Choice Question Set IV Answers
31-b, 32-d, 33-d, 34-b, 35-b, 36-b, 37-a, 38-c, 39-c, 40-b

Multiple Choice Question Set V

41) Excess Carbon dioxide (CO2) found in
a) Metabolic acidosis
b) Metabolic alkalosis
c) Respiratory acidosis
d) Respiratory alkalosis

42) Maple syrup disease is disorder due to deficiency of pathway that degrades:
a) Tyrosine
b) Tryptophan
c) Leucine
d) Methionine

43) Amine groups of protein are excreted in urine as:
a) Amino acids
b) Creatine
c) Urea
d) Ammonia

44) …is the hormone, predominantly produced in response to stress and use cAMP as the second messenger.
a) Thyroxine
b) Parathyroid Hormone (PTH)
c) Epinephrine
d) Calcitonin

45) One of the following hormones is an amino acid derivative:
a) Thyroxine
b) ADH
c) Cortisol
d) Prolactin

46) The PIP3-derived second messenger that mobilizes intracellular calcium is:
a) PIP2
b) I-1,4,5-P3
c) Phosphatidic acid
d) DAG

47) A 14-year old boy comes to the outpatient clinic complaining of increased thirst and tiredness. His random blood glucose level is 234.0 mg/dL ( 13.0 mmol/L).Would you:
a) Repeat the random plasma glucose measurement?
b) Perform fasting plasma glucose measurement?
c) Perform the OGTT?
d) Measure urine ketones?

48) Thyrotropin-releasing hormone(TRH) stimulates the secretion of:
a) Prolactin
b) TSH
c) PTH
d) Luteinizing hormone

49) Hypothyroidism is generally associated with all of the following EXCEPT:
a) Weight loss
b) TPO antibodies
c) An elevation of TSH levels
d) TSH-receptors antibodies

50) The biochemical investigation of the blood specimen showed Bilirubin- 4.5 mg/dl, ALT- 150 IU/L, AST- 200 IU/L, Albumin- 25 mg/dL. What is the likely biochemical diagnosis?
a) Hemolytic Jaundice
b) Acute hepatic disease
c) Chronic hepatic disease
d) Obstructive liver disease

Multiple Choice Questions Set V Answers
41-d, 42-c, 43-c, 44-c, 45-a, 46-b, 47-b, 48-b, 49-a, 50-c

Multiple Choice Question Set I

1) Which of the following enzyme is responsible for detoxification of the intermediates produced during O2 reduction to H2O?
a) Oxygen reductase
b) Catalase
c) Cytochrome C
d) Cytochrome oxidase

2) The Km is the concentration of substrate when Vo is
a) ¼ of Vmax
b) 1/2 of Vmax
c) 1/3 of Vmax
d) 2/3 of Vmax

3) Identify the oxidized form of co-enzyme form the following:
a) FADH
b) NADH
c) NADPH
d) NADP

4) Identify an allosteric enzyme from the following:
a) fructokinase
b) phosphofructokinase
c) fructose 1,6 bisphosphatase
d) glucokinase

5) Which one of the followings is not true about genetic code?
a) It is degenerate
b) It is unambiguous
c) It is nearly universal
d) It is overlapping

6) CAAT box is present in many
a) Prokaryotic promoters upstream of TATA box.
b) Prokaryotic promoters that are downstream of TATA box.
c) Eukaryotic promoters that are upstream of TATA box.
d) Eukaryotic promoters that are downstream of TATA box.

7) Negative supercoils are introduced in DNA by
a) Helicase
b) DNA ligase
c) DNA gyrase
d) DNA polymerase III holoenzyme

8) A ten-year-old girl is brought to the dermatologist by her parents. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Which of the following processes is most like to be defective in this patient?
a) Removal of primers from Okazaki fragments
b) Removal of mismatched bases from the 3' end of Okazaki fragments
c) Removal of pyrimidine dimers from DNA
d) Removal of uracil from DNA

9) DNA polymerase III holoenzyme possesses:
a) polymerase activity only
b) 3’→ 5’ endonuclease activity
c) 3’→ 5’ exonuclease activity and polymerase activities
d) 5’→ 3’ exonuclease activity

10) Cyclins:
a) are circular DNA double helices
b) are circular polypeptides
c) are proteins that regulate cell cycle
d) are proteins that induce apoptosis

Multiple Choice Questions Set I Answers
1-b, 2-b, 3-d, 4-d, 5-d, 6-a, 7-c, 8-c, 9-c, 10-c

Adeno associated virus (AVV) is a naturally occurring single-stranded DNA virus that belongs to the parvovirus family. The commonly used viral vectors for gene therapy are derived from naturally occurring AVVs. These vectors are genetically modified/engineered that do not contain genes encoding viral proteins such as rep and cap. These genes are replaced by the gene of interest and transduced into humans for the treatment of specific diseases. AAV viral vectors enable attachment and entry of transgene into the target cells. Once inside the cell nucleus, the transgene remains in the episomal form and expresses the gene of interest. Although recent evidence has shown that long-term stable expression of these transgenes can be achieved in pre-clinical models and humans, there is inherent immunogenicity risk associated with adeno-associated vectors which include:
-Pre-existing antibodies and T cell response against viral vectors
-Neutralizing antibodies generated against capsid proteins of the adeno-associated virus vector
-T cell-mediated response against AVV vector capsid
-Neutralizing antibodies and T cell-mediated response against transgenes



Risk and Consequences of immunogenicity against adeno-associated virus-mediated gene therapy


Pre-existing antibodies and T cell response against viral vectors: The high prevalence of pre-existing binding and neutralizing antibodies against adeno-associated viral vectors are observed in clinical studies. These are plausibly cross-reactive antibodies generated against wild-type adeno-associated viruses. Seroprevalence studies have shown that anti-AAV2 neutralizing antibodies display the highest prevalence followed by anti-AVV1 and neutralizing antibodies against other AVV serotypes such as AAV7, AAV8, and AAV9. The seroprevalence of antibody varies with geographical regions and direct comparison of prevalence studies is limited by different detection methods and sensitivity.
In case of T cell-mediated immunity, the stimulation of a maximal adaptive immune response requires co-stimulation with the helper virus leading to CD4+ and CD8+ T cells directed to the antigens of the helper virus and of the adeno-associated virus are formed, and a small pool of memory T cells are maintained throughout life.
Neutralizing antibodies generated against capsid proteins of adeno-associated virus vector: The AVV vectors are genetically modified to ensure that it does not integrate into the host genome. This has decreased risk of random integration, gene disruption or cancer. But the capsid proteins are intact viral proteins that are recognized by the host immune cells activating immune response and seroconversion. The gene therapy involves a single large dosage of gene-containing vector particles which may provoke rapid innate and adaptive immune response. The immunogenicity and antibody response to the viral capsid can vary from individual to individual and the mechanism of this variation is largely unknown. But the neutralizing antibodies generated against these viral particles have shown to decrease viral vector delivery to the target cell by increasing the clearance of viral particles.
An alternate approach to attain the optimal transduction for sufficient expression of transgene would be to increase the dose of the viral particle which has higher risk immunogenicity response and toxicity. In a few studies, tapered glucocorticoid administration after seroconversion has shown to improve the transduction and stable transgenesis. But there is conflicting evidence on the stable expression of transgene after the withdrawal of immunosuppressants, even though tissue biomarker for tissue injury has returned to with normal reference interval.
T cell-mediated response against AVV vector capsid: Initially, T cell-mediated immune response was observed in hemophilia B subjected who was given engineered AVV2 vector consisting of factor IX under a liver-derived promoter. In a study subject, approximately 4 weeks after vector infusion, the liver transaminases (alanine aminotransferase, aspartate aminotransferase) began to rise, peaking at about 6 weeks and gradually returning to normal levels over the ensuing 8 weeks without any medical intervention. Concurrently, circulating factor IX (FIX) levels also slowly declined and by 12 weeks after vector infusion was decreased to pretreatment baseline. IFN-gamma enzyme-linked immunospot (ELISPOT) assays showed a response to AAV2 capsid but not transgene (factor IX). It was hypothesized that the transaminase elevation reflected the specific destruction of the transduced hepatocytes by capsid-directed CD8+ T cells. Another observation from different clinical studies has shown that tapered treatment of glucocorticoids (immunosuppressant) would decrease elevated transaminases to baseline and partially rescue the loss of transgene. Therefore, current clinical trials have incorporated the co-infusion of immunosuppressants with viral particles increases transduction and decrease immune response. In a recent study, the stable transgene expression is observed even withdrawal of immunosuppressants after liver enzymes return to baseline in transaminases elevated subjects.
Neutralizing antibodies and T cell-mediated response against transgenes: The neutralizing antibodies against viral proteins may inhibit the transduction of genetically engineered viral particles into the host cells. In contrast, the neutralizing antibodies against the transgenes can have a greater risk to the host including antibody-mediated and T-cell mediated immunity. Although, to date, no neutralizing antibodies and T-cell-mediated immune responses are observed, these trials are conducted in an immune-privileged organ or the inclusion of subjects that do not have pre-existing neutralizing antibodies. Results from clinical trials are concordant with results from preclinical models where immune tolerance has been observed and are mainly associated with the induction of Tregs (T regulatory cells). These results are yet to be translated into humans and a broader study should be conducted to understand the full spectrum of immune response and immune tolerance.