What is reperfusion injury? Discuss the mechanism of reperfusion injury.

Reperfusion injury:

Restoration of blood flow to ischemic tissues can promote recovery of cells if they are reversibly injured, but  can also paradoxically exacerbate the injury and cause cell death.

Mechanism:

The likely answer is that new damaging processes are set in motion during reperfusion, causing the death of cells that might have recovered otherwise. Several mechanisms have been proposed.

• Oxidative stress. New damage may be initiated during reoxygenation by increased generation of reactive oxygen and nitrogen species.
• Intracellular calcium overload. Intracellular and mitochondrial calcium overload  begins during acute ischemia; it is exacerbated during reperfusion due to influx of calcium resulting from cell membrane damage and ROS mediated injury to sarcoplasmic reticulum.
• Inflammation. Ischemic injury is associated with inflammation as a result of “dangers signals” released from dead cells, cytokines secreted by resident immune cells such as macrophages, and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells, all of which act to recruit circulating neutrophils to reperfused tissue.
• Activation of the complement system may contribute to ischemia-reperfusion injury.

Write down the mechanism of irreversible cell injury.

Mechanisms of cell injury are as follows:

• Depletion of ATP: Reduction in ATP levels is fundamental cause of necrotic cell death. ATP depletion and decreased ATP synthesis are frequently associated with both hypoxic and chemical injury. The  major causes of ATP depletion are reduced supply of oxygen and nutrients, mitochondrial damage, and the actions of some toxins.
• Mitochondrial Damage: Mitochondria are critical players in cell injury and cell death by all pathways. Mitochondria can be damaged by increases of cytosolic Ca2+, reactive oxygen species, oxygen deprivation and mutations in mitochondrial genes in some inherited diseases.
• Influx of Calcium and Loss of Calcium Homeostasis: Calcium ions are important mediators of cell injury.
• Accumulation of Oxygen-Derived Free Radicals: Cell injury induced by free radicals, particularly reactive oxygen species, is an important mechanism of cell damage in many pathologic conditions, such as chemical and radiation injury, ischemia-reperfusion injury, cellular aging, and microbial killing by phagocytes.
• Defects in Membrane Permeability: Early loss of selective membrane permeability, leading ultimately to overt membrane damage, is a consistent feature of most forms of cell injury. Membrane damage may affect the functions and integrity of all cellular membranes.
• Damage to DNA and Proteins.

What are the morphologic changes in cell injury.

Morphologic changes in reversible cell injury:

Light microscopy:

Cellular swelling is the first manifestation of almost all forms of injury to cells. It causes some pallor, increased turgor, and increase in weight of the organ. On microscopic examination, small clear vacuoles may be seen within the cytoplasm; these represent distended and pinched-off segments of the ER. This pattern of nonlethal injury is sometimes called hydropic change or vacuolar degeneration. Cells may also show increased eosinophilic staining, which becomes much more pronounced with progression to necrosis. 

The ultrastructural changes of reversible cell injury include: 

1.Plasma membrane alterations, such as blebbing, blunting, and loss of microvilli 

2.Mitochondrial changes, including swelling and the appearance of small amorphous densities 

3.Dilation of the ER, with detachment of polysomes; intracytoplasmic myelin  figures may be present 

4.Nuclear alterations, with disaggregation of granular and fibrillar elements.

Consequences of Hypoxic injury

Consequences of hypoxic injury:

• As the oxygen tension within the cell falls, there is loss of oxidative phosphorylation and decreased generation of ATP. The depletion of ATP results in failure of the sodium pump, leading to efflux of potassium, influx of sodium and water, and cell swelling. 
• There is also influx of Ca2+, with its many deleterious effects. 
• There is progressive loss of glycogen and decreased protein synthesis. The functional consequences may be severe at this stage.
• The cytoskeleton disperses, resulting in the loss of ultrastructural features such as microvilli and the formation of blebs at the cell surface. 
• Myelin figures derived from degenerating cellular membranes, may be seen within the cytoplasm or extracellularly. They are thought to result from unmasking of phosphatide groups, promoting the uptake and intercalation of water between the lamellar stacks of membranes. 
• At this time the mitochondria are usually swollen, as a result of loss of volume control in these organelles; the ER remains dilated; and the entire cell is markedly swollen, with increased concentrations of water, sodium, and chloride and a decreased concentration of potassium. 
• If ischemia persists, irreversible injury and necrosis ensue.

Describe briefly the sources and possible consequences of increased cytosolic calcium in cell injury.

Sources of calcium:

1. Intracellular source: Mitochondria, Endoplasmic reticulum.

2. Extracellular source.

Consequences of increased cytosolic calcium:

• The accumulation of Ca2+ in mitochondria results in opening of the mitochondrial permeability transition pore and failure of ATP generation. 
• Increased cytosolic Ca2+ activates a number of enzymes with potentially deleterious effects on cells. These enzymes include phospholipases (which cause membrane damage), proteases (which break down both  membrane and cytoskeletal proteins), endonucleases (which are responsible for DNA and chromatin  fragmentation), and ATPases (thereby hastening ATP depletion).
• Increased intracellular Ca2+ levels also result in the induction of apoptosis, by direct activation of caspases and by increasing mitochondrial permeability.

Describe the biochemical mechanism leading to cell membrane damage.

Mechanisms of cell Membrane Damage:

• Reactive oxygen species. Oxygen free radicals cause injury to cell membranes by lipid peroxidation.
• Decreased phospholipid synthesis. The production of phospholipids in cells may be reduced as a consequence of defective mitochondrial function or hypoxia, both of which decrease the production of ATP and thus affect energy-dependent biosynthetic pathways. The decreased phospholipid synthesis may affect all cellular membranes, including the mitochondria themselves.
• Increased phospholipid breakdown. Severe cell injury is associated with increased degradation of membrane phospholipids, probably due to activation of calciumdependent phospholipases by increased levels of cytosolic and mitochondrial Ca2+.
• Cytoskeletal abnormalities. Cytoskeletal filaments serve as anchors connecting the plasma membrane to the cell interior. Activation of proteases by increased cytosolic calcium may cause damage to elements of the cytoskeleton.

What are the morhological types of irreversible cell injury?

Morphological pattern of irreversible cell injury are as follows: 

A. Necrosis

• Coagulative necrosis
• Liquefactive necrosis
• Gangrenous necrosis
• Caseous necrosis
• Fat necrosis
• Fibrinoid necrosis

B. Apoptosis.

What are the causes of cell injury?

Causes of Cell Injury: 

The causes of cell injury range from the external gross physical violence of an automobile accident to subtle internal abnormalities, such as a genetic mutation causing lack of a vital enzyme that impairs normal metabolic function. Most injurious stimuli can be grouped into the following broad categories.

1. Oxygen Deprivation. Causes of hypoxia include reduced blood flow (celled ischemia), inadequate oxygenation of the blood due to cardiorespiratory failure, and decreased oxygen-carrying capacity of the blood, as in anemia or carbon monoxide poisoning (producing a stable carbon monoxyhemoglobin that blocks oxygen carriage) or after severe blood loss.

2. Physical Agents. Physical agents capable of causing cell injury include mechanical trauma, extremes of temperature (burns and deep cold), sudden changes in atmospheric pressure, radiation, and electric shock.

3. Chemical Agents and Drugs. Oxygen at high concentrations is toxic, Arsenic, Cyanide, Mercuric salts.

4. Infectious Agents. Rickettsiae, Bacteria, Fungi, and higher forms of parasites.

5. Immunologic Reactions. Injurious reactions to endogenous self-antigens are responsible for several autoimmune diseases.

6. Genetic Derangements.

7. Nutritional imbalance. Protein-calorie deficienciency, Deficiencies of specific vitamins, Atherosclerosis, Obesity.

What is cell injury? What are the types of cell injury?

Cell injury:

When cells are stressed so severely that they are no longer able to adapt or when cells are exposed to inherently damaging agents or suffer from intrinsic abnormalities. Injury may progress through a reversible stage and culminate in cell death.

Types of cell injury:

1. Reversible cell injury.

2. Irreversible cell injury or cell death.

Isoelectric pH.

• It is the pH at which biomolecules exist as dipolar ion possessing same amount of positive and negative charge on their surface with net zero.
• Biomolecules contain different ionizable groups on their surface.
• Depending on the pH of solution these ionizable groups act either as proton donor or as proton acceptor and thereby express charge on their surface.
• At one definite pH called isoelectric pH, every biomolecule expresses both the charges on its surface in equal amount and exists as zwitter ion, when the net charge on the surface of biomolecule is zero because positive and negative charge equalize each other. So zwitter ions are electrically neutral without any electrohoretic mobility during electrophoresis.
• At pH below the isoelectric pH biomolecules accept protons from the medium and exist as cations with positive charge on their surface and at pH above the isoelectric pH biomolecules release proton to the medium and exist as anions with negative charge on their surface. 

pH of a solution.

P^H is the negative logarithm of hydrogen ion concentration of a solution, when H⁺ concentration is expressed in terms of molarity

 

[H+] in mol/L	Calculation of pH	pH
10-7	-log 10-7	7
10-8	-log 10-8	8
10-3	-log 10-3	3

What is free acidity and what is titratable acidity.

Free acidity:

• Free acidity of a solution is its instant H ion concentration measured without any manipulation.
• Free equimolar concentration of strong acid and weak acid, the free acidity will be very high in strong acid solution compared to that in weak acid solution because strong acids are completely dissociated and weak acids are partially dissociated into hydrogen ions.

Titratable acidity:

• Titratable acidity of a solution is its total H ion concentration that would be if the acid was allowed to ionize totally to hydrogen ion following its titration with alkali.
• For equimolar concentration of strong acid and weak acid the total hydrogen ion concentration of both the solution will be same at the end of complete ionization following titration with alkali.
• Very small amount of weak acid usually ionize to hydrogen ion and majority fail to ionize. During titration when alkali is added to the medium, the hydrogen ion concentration of the medium decreases which facilitate the unionized weak acid to ionize completely to hydrogen ion.
• Equimolar concentration of strong acid and weak acid differ with respect to free acidity, but they are same the respect to the titratable or total acidity.        

What is Amphoteric substance?

These are the substances which can act both as acid as well as base in different situation.

Properties of alkaali.

• Alkali are metallic hydroxides of alkaline metals, which in solution ionize to hydroxyl ions that can bind H to form H2O and thereby can remove hydrogen ion from solution.
• Strong alkalis are those which rapidly and almost completely ionize to hydroxyl ions that quickly remove hydrogen ion from solution.
• Weak alkalis are those which slowly and partially ionize to hydroxyl ions that slowly remove hydrogen ion from solution.
• In fact all alkalis are also regarded as base, but all bases are not alkalis. 

Properties of Base.

• Bases are proton acceptor in aqueous solution.
• Bases may be charged particle or may be without charge.
• Strong bases are those which have greater tendency to accept proton. They bind rapidly and strongly with strongly with proton, so quickly remove from solution.
• Weak bases are those which have very low tendency to accept proton. They bind slowly and weakly with proton, so slowly remove proton from solution.
• Conjugate acid of a base is the acid formed by that base after accepting proton. So conjugate acid of a strong base is weak and that of a weak base is strong.

Properties of Acid.

• Acids are proton donor in aqueous solution.
• Acids are molecules having hydrogen atom and capable to release hydrogen ion in aqueous solution.
• Acids may be the molecular species with positive charge or negative charge or may be without charge.
• Conjugate base of an acid is the remaining anionic part of an acid after removal of proton from that acid.
• Strong acid is the acid which rapidly and completely ionizes into H+ and its conjugate base in solution.
• Weak acid the acid which slowly and partially ionizes into hydrogen ion and its conjugate base in solution.
• For strong acid degree of dissociation and dissociation constant is high with low pK value. Conversely for weak acid degree of dissociation and dissociation constant is low with high pK value.
• In case of strong acid the conjugate base shows less affinity to proton because of which they can rapidly and completely ionize to proton. So conjugate bases of strong acids are weak in nature. On the other hand, in case of weak acid the conjugate base shows strong affinity to proton because of which they partially ionize to proton. So conjugate bases of weak acids are strong in nature. 

Radiation hazard

Radiation hazards are as follows:

1. Immediate hazards
• Bone marrow depression
• Immune suppression
• Damage to intestinal mucosa causing diarrhea and malabsorption.
• Baldness
• Rough and scaly skin
• In pregnancy: Fetal growth retardation, congenital malformations of fetus, fetal death.
2. Delayed hazards
• Carcinogenesis
• Sterility
• Cataract
3. Genetic defects
• DNA damage
• Mutation

Name the radio-sensitive tissue.

The following tissues are sensitive to radiation. These are mostly rapidly dividing tissues.

1. Bone marrow
2. Gonads
3. Lymph node
4. Skin
5. Intestine.

What is radioactivity? Clinical use of radioactive isotope.

Radioactivity:

It is the spontaneous emission of accelerated particles(radiation) from an unstable isotope by radioactive decay.

Clinical use of radio active isotope:

A. Diagnostic use

1. Iodine uptake test for diagnosis of thyroid disorders.
2. Radio immune assay of hormones for diagnosis of hormone disorders.
3. Organ scanning e.g. bone scan, brain scan, thyroid scan.
4. Absorption test e.g. for iron. vitamin B12
5. Isotope renogram for mesurement of GFR and renal clearance.
6. RBC life span measurement.

B. Therapeutic use, e.g. radiotherapy in treatment of malignancy.

C. Use in tracer technique: isotopes are used as tracer in metabolic studies to outline the metabolic pathways.

D. Measurement of volumes and spaces. e.g. ECF volume, Blood volume, plasma volume, RBC volume.

E. Measurement of regional blood flow. e.g. Cerebral blood flow, coronary blood flow, renal blood flow.

F. Sterilization of medical instruments.

Isotope: Definition, Types.

Definition:

• Atoms with same atomic number (number of proton), but different atomic weight (number of protons and neutrons).
• Atoms of same element with different atomic weight.

Types of isotope:

1. Stable isotope
• Stability of an isotope depends on the definite neutron to ration which is specific for a specific atom.
• In atoms of low atomic weight stability is usually achieved with neutron to proton ration around one.
• In atoms of high atomic stability is usually achieved with more neutron than proton
• Neutrally occurring isotopes of most of the predominant elements are stable isotopes.
2. Unstable isotope
• These are the isotopes having neutron to proton ration far away from its stability ratio.
• Neutrally occurring isotopes of heavy elements are usually unstable
• Rarely some naturally occurring isotopes of lighter elements can also be unstable
• Unstable isotopes tend to become stable by radio active decay

What is Epimer?

Epimer:

Epimers are the optical stereoisomers which differ with respect to the spatial configuration around only one asymmetric carbon out of more than one asymmetric carbon in the molecule.

 e,g.  Glucose and Mannose (they are C2 epimers)
         Glucose and Galactose (they are C4 epimers)

Isomer: Definition, Types.

Isomer:

Definition:

Isomers are substances having same molecular (chemical) formula, but different structure or having
same molecular formula and identical structural form, but different spatial configuration around one
or more carbon. Isomerism is the processes of formation of isomers.

Types of isomer:

1. Structural isomer
2. Stereo isomer (Space isomer)
• Geometric isomer
• Cis variety
• Trans variety

•  Optical isomer
• Optical enantiomers
• Diasteroisomers

Structural Isomer:

These are the substances having same molecular (chemical) formula, but different structure. These
type of isomers differ with respect to physical and chemical properties.
e.g.     CH3-CH2-CH2-CH3 (Butane)
           
                     CH3

           CH3-CH-CH3 (Isobutane)

Stereo Isomer (Space Isomer)

Substances having same molecular formula as well as identical structural form, but different with
respect to the spatial configuration of atoms or groups around one or more carbon. This type of
isomers sometimes shows identical physical and chemical properties.
Spatial configuration means
o Arrangement of atoms or groups around a carbon in relation to space,
o Three dimensional space relationship of atoms or groups around a carbon.

Types of stereo isomer:

1. Geometric isomer
2. Optical isomer
• Optical enantiomers
• Diastereoisomers

Chronic liver disease

Introduction:

·         Primary care physicians play a key role in early identification of risk factors, in the management of patients for improving quality and length of life, and for preventing complications.

·         Specialists, by contrast, should guide specific treatments, especially in the case of complications and for selecting patient candidates for liver transplantation.

Etiology:

·         Hepatitis B

·         Hepatitis C

·          Non Alcoholic Steato Hepatitis(NASH)

·         Alcohol abuse

·         Others e.g. Primary biliary cirrhosis, α₁-antitrypsin deficiency, Hemochromatosis, Wilson disease, cryptogenic.

Clinical Features:

·         Often asymptomatic

·         May present with stigmatas of CLD such as vascular spiders, palmar erythema, spider angiomata, palmar erythema, Gynaecomastia, dilated abdominal veins, loss of hair, testicular atrophy.

·          jaundice, ascites, splenomegaly and asterixis  indicate signs of decompensation

·         Patients may be diagnosed incidentally through laboratory findings

Investigations:

• ·         CBC
• ·         Liver function tests ( S. Bilirubin , SGOT, SGPT, ALP, S.albumin, A: G Ratio, Prothrombin Time)
• ·         S. electrolytes,
• ·         S. creatinine
• ·         USG of whole abdomen—coarse liver, splenomegaly, ascites
• ·         Viral Marker ( HBS Ag, Anti HCV)

Management:

A. General

Nutrition, Fluid and Electrolyte:

• Diet according to patient’s status eg.  Malnourished (high protein and carbohydrate), hepatic encephalopathy/precoma (Protein restriction) Ascites (salt restriction).
•  Lactulose in constipation (20 – 30 ml/day)
• Stop OCP, Sedatives, NSAID and Paracetamol.

 B. Management according to presentation:

Ascites

• ·         No added salt,
• ·         Paracentesis  for diagnosis and therapy:
•           Send sample for biochemistry/cytology and C/S if possible.
• ·         Spironolactone (100 mg - 400 mg daily) and/or furosemide (40 mg - 160 mg daily),
•           Measure weight  daily, target weight loss at ~500g/day.
•           The dose of diuretics - increased every 3–4 days to achieve target weight loss.
• ·         For spontaneous bacterial peritonitis (SBP), based on abdominal pain, fever, and ascitic fluid report.
•            Start IV Cefotaxim 1g tds/ ciprofloxacin 200mg IV BD for 7 - 10 days.
•           Antibiotic  prophylaxis  included ciprofloxacin ,750 mg orally once weekly
• ·         If there is massive ascites –   Refer to secondary level/ Tertiary level

Hepatic Encephalopathy  

Grade I: Changes in behaviour with minimal change in level of consciousness

Grade II: Disorientation, drowsiness, asterixis, inappropriate behaviour

Grade III: Marked confusion, incoherent speech, sleeping but rousable

Grade IV: Comatose, unresponsive, decorticate or decerebrate posturing

• ·         Avoid sedatives/ diuretics
• ·         Maintenance of Nutrition and fluid  balance
• ·         Lactulose 20mL tds (titrate dose to achieve at least 2 loose stools/day),  if necessary enema until 2–4 bowel movements/day .
• ·         Amoxicillin (500mg TDS)/ Metronidazole (400mg TDS)/Rifaximin (400 mg TDS) for gut sterilization.
•      Grade 3 or 4 encephalopathy-Refer           

Hematemesis and Melaena

·         I/V Fluid (N/S, Hartman sol.), Immediate blood transfusion and refer.                                                                                                                                                                                                                  

 Coagulopathy  Characterized by  prolonged prothrombin time

·           Vitamin K - 10mg I/V for 3 days.

Severe coagulopathy, Vit K can be given IV 10mg slowly and Refer

Hepatorenal syndrome – Characterized by oliguria in cirrhotic patient without proteinuria and abnormal sediment in urine.

·         Refer.

·         Before referral--   Stop  diuretics & NSAIDs and Catheterise bladder

Indication for Referral:

1.  Decision for antiviral therapy   2.  Variceal   haemorrhage    3.  Massive ascites                4.  Diagnostic and  therapeutic   Endoscopy. 5.  Encephalopathy Grade III & IV                    6.  Hepatorenal syndrome  7. Multi-organ failure   8.  Hepatocellular carcinoma.