CASE STUDIES
✓ Case Study 1
A 62-year-old man suffered from congestive heart failure. He was hospitalized and was started with a loading dose of digitalis. Gross improvement was seen in his symptoms. He was discharged after a few days and was put on a maintenance dose of digitalis.
What is the chemical nature of digitalis?
How does it help in improving the symptoms of heart failure?
CASE DISCUSSION
Cardiac failure can be described as the inability of the heart to pump blood effectively at a rate that meets the needs of the metabolizing tissues. Cardiac failures primarily arise from the reduced contractility of heart muscles, especially the ventricles. Reduced contraction of heart leads to reduced cardiac output resulting in the increase in cardiac blood volume. The heart feels congested. Hence the term congestive heart failure.
Congested heart leads to hypotension and poor renal perfusion. This results in the development of edema in the lower extremities, pulmonary edema as well as renal failure.
Cardiac glycosides are used for the treatment of congestive heart failure and arrhythmias. Digitalis used in the given case is a cardiac glycoside.
BASIC CONCEPT
Glycosides are compounds containing a carbohydrate and a non-carbohydrate residue in the same molecule. In these compounds the carbohydrate residue is attached by an acetal linkage of carbon-I- to the non-carbohydrate residue. The non-carbohydrate residue present in the glycoside is called as Aglycone. The aglycones present in the glycosides vary in complexity from simple substances as Methyl Alcohol, glycerol, phenol or a base such as Adenine to complex substances such as sterols, hydroquinones and anthraquinones. The glycoside is named according to the carbohydrate it contains. If it contains Glucose then it forms and if it contains galactose, then it forms galactoside, etc.
Glycosides are present in many drugs, spices and in the constituents of animal tissues.
Cardiac Glycosides
Cardiac glycosides all contain steroids as aglycone or genin component in combination with sugar molecules. These include derivatives of Digitalis and Strophanthus such as oubain.
Aglycone: The steroid nucleus has a unique set of fused ring system that makes the aglycone moiety structurally distinct from the other more common steroid ring systems. The steroid nucleus has hydroxyls at 3- and 14- positions of which the sugar attachment uses the 3-OH group. 14-OH is normally unsubstituted.
Sugar: One to four sugars are found to be present in most cardiac glycosides attached to the 3-OH group. The sugars most commonly used include L-rhamnose, D-glucose, D-digitoxose, D-digitalose, D-digginose, D-sarmentose, L-vallarose, and D-fructose. These sugars predominantly exist in the cardiac glycosides in the β-conformation.
The pharmacological activity is contained in the aglycone, which has however a less potent and shorter action than the parent glycoside. The sugar ensures increase water solubility, cell penetrability and potency of the aglycone.
- Digitalis: Digitalis was previously the drug of choice for treating congestive heart failure and arrhythmias. but its use in patients who are in sinus rhythm has declined since the therapeutic-to-toxic ratio is quite narrow.This is a purified glycoside from European foxglove, Digitalis purpurea.A group of pharmacologically active compounds are extracted mostly from the leaves in pure form and are referred to by common chemical names such as digitoxin or digoxin.
Mechanism of Action of Digitalis
Direct action: The most probable mechanism of action for the positive inotropic effect of digitalis is inhibition of the membrane-bound Na+/K+ - ATPase pump directly which acts as the digitalis receptor; when this occurs, Na+ increases in the cell, the exchange of Na+ for Ca2+ is augmented, and calcium influx is increased. The increased intracellular calcium in turn leads to increased release of Ca2+ from the sarcoplasmic reticulum and increased contractility of the cardiac muscle. Changes in the ratio of intracellular and extracellular electrolytes can result in increased automaticity and cardiac arrhythmias. The inhibition of the Na+/K+ - ATPase pump prevents the re-entry of potassium into the cell after the repolarization is complete, leading to depletion of the intracellular potassium. This potassium depleting action of the digitalis is not confined to the cardiac muscle but also involves the skeletal muscle and the liver. By direct action it increases the force of contraction of the heart muscle.
Digitalis also has a negative chronotropic effect due to decreased conduction velocity in the atrioventricular (AV) node.
Therapeutically it is very useful in heart failure associated with atrial fibrillation and rapid ventricular rate.
- Oubain: A glycoside obtained from Strophanthus sp. Inhibits active transport of Na+ in the cardiac muscle (Sodium pump Inhibitor). It also acts as a cardiac glycoside. It is now rarely used.
Other Glycosides of Clinical Importance
Many biologically active compounds are glycosides. Glycosides comprise several important classes of compounds such as hormones, sweeteners, alkaloids, flavonoids, antibiotics, etc. The glycosidic residue can be crucial for their activity or can only improve pharmacokinetic parameters.
Other glycosides such as streptomycin are used as antibiotics. Phloridzin is another glycoside which is obtained from the root and bark of apple tree. It blocks the transport of sugar across the mucosal cells of small intestine and also renal tubular epithelium. It displaces Na+ from the binding site of “carrier protein” and prevents the binding of sugar molecule and produces glycosuria.
Glycosides of vitamins, both hydrophilic and lipophylic, often occur in nature. Glycosylated vitamins have an advantage over the respective aglycone in their better solubility in water (especially the lipophylic ones), stability against UV-light, heat and oxidation, reduction of the bitter taste and odor (e.g. thiamine), and resistance to an enzymatic action. Some of the vitamin glycoconjugates have altered or improved Pharmacokinetic properties.
Clinical Note
The inhibition of the Na+/K+ - ATPase pump by digitalis prevents the re-entry of potassium into the cell after the repolarization is complete; this causes depletion of the intracellular potassium.
Clinical Pearls
- Glycosides are compounds containing a carbohydrate and a non-carbohydrate residue in the same molecule.
- Cardiac glycosides all contain steroids as aglycone component in combination with sugar molecules.
- Glycosides comprise several important classes of compounds such as hormones, sweeteners, alkaloids, flavonoids, antibiotics, etc.
- The most probable mechanism of action for the inotropic effect of digitalis is inhibition of the membrane-bound Na+/K+ - ATPase pump.
- Therapeutically it is very useful in heart failure associated with atrial fibrillation and rapid ventricular rate.
✓ Case Study 2
A 54-year-old woman who was bed bound in a nursing home began to develop swelling of her left leg. She was evaluated with venous Doppler ultrasound and was found to have a deep vein thrombosis. She was immediately started on heparin to prevent the clot from further enlarging.
What is the chemical nature of heparin?
How will it help in preventing the clot formation?
CASE DISCUSSION
Heparin is a highly sulfated GAG (Glycosaminoglycan) see the details below:
Heparin
Heparin is also called α Heparin. It is an anticoagulant widely used in clinical practice. It is present in liver, lungs spleen and monocytes. Commercial preparations are from animal lung tissues. It contains repeating units of sulphated glucosamine and either of the two uronic acids-D-glucuronic acid and L-Iduronic acid. In fully formed heparin molecules 90 percent or more of uronic acid residues are L-Iduronic acid. It is strongly acidic due to sulphuric acid groups and readily forms salts.
Clinical Role of Heparin
In vitro Heparin is used as an anticoagulant while taking blood samples, 2 mg/10 ml of blood is used. It is considered the most satisfactory anti-coagulant as it does not produce a change in red cell volume or interfere with subsequent determinations.
In vivo Heparin is used in suspected thromboembolic conditions to prevent intravascular coagulation. Heparin is used for anticoagulation for the following conditions:
- Acute coronary syndrome,
- Atrial fibrillation
- Deep-vein thrombosis and pulmonary embolism (Heparin and its derivatives enoxaparin, dalteparin, tinzaparin are effective at preventing deep-vein thromboses and pulmonary emboli in patients at risk).
- Cardiopulmonary bypass for heart surgery.
- ECMO circuit for extracorporeal life support.
Mechanism of Action
Role of heparin as an anticoagulant: The biological effect of heparin is to bind and activate antithrombin, which in turn inactivates thrombin and factor X. Binding of Heparin to lysine residues in antithrombin III produces conformational changes which promote the binding of the latter to serine protease “thrombin” which is inhibited, thus fibrinogen is not converted to fibrin and the coagulation is inhibited.
Role of heparin as a coenzyme: Heparin acts in the body to potentiate the activity of the enzyme “Lipoprotein lipase”. Heparin binds specifically to the enzyme present in capillary walls causing its release into the circulation. Hence it is also called releasing factor.
Administration of Heparin
Heparin is given parenterally, as it is degraded when taken by mouth. It can be injected intravenously or subcutaneously. Intramuscular injections are avoided because of the potential for forming hematomas.
Because of its short biologic half-life of approximately one hour, heparin must be given frequently or as a continuous infusion. However, the use of low-molecular-weight heparin (LMWH) has allowed once-daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until the oral anticoagulant Warfarin takes effect.
Adverse Effects
A serious side effect of heparin is heparin-induced thrombocytopenia (HIT). HIT is caused by an immunological reaction that makes platelets a target of immunological response, resulting in the degradation of platelets. This is what causes thrombocytopenia. This condition is usually reversed on discontinuation, and can generally be avoided with the use of synthetic heparins. There is also a benign form of thrombocytopenia associated with early heparin use, which resolves without stopping heparin.
There are two nonhemorrhagic side effects of heparin treatment. The first is elevation of serum Aminotransferases levels, which has been reported in as many as 80 percent of patients receiving heparin. This abnormality is not associated with liver dysfunction, and it disappears after the drug is discontinued. The other complication is hyperkalemia, which occurs in 5 to 10 percent of patients receiving heparin, and is the result of heparin-induced aldosterone suppression. The hyperkalemia can appear within a few days after the onset of heparin therapy.
Clinical Note
In vivo heparin is used in suspected thromboembolic conditions to prevent intravascular coagulation.
Clinical Pearls
- Heparin is component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin.
- It contains repeating units of sulphated glucosamine and either of the two uronic acids-D-Glucuronic acid and L-Iduronic acid.
- The biological effect of heparin is to bind and activate antithrombin, which in turn inactivates thrombin and factor X.
- Heparin binds specifically to the lipoprotein lipase enzyme present in capillary walls causing its release into the circulation. Hence it is also called releasing factor.
- Heparin is given parenterally, as it is degraded when taken by mouth.
BASIC CONCEPT
Glycosaminoglycans
The most abundant heteropolysaccharide in the body are the glycosaminoglycans (GAGs). GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution. GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM). Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides passageways between cells, allowing for cell migration.
The specific GAGs of physiological significance are hyaluronic acid, dermatan sulfate, Chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate. Although each of these GAGs has a predominant disaccharide component, heterogeneity does exist in the sugars present in the make-up of any given class of GAG. These molecules are long unbranched polysaccharides containing a repeating disaccharide unit. The disaccharide units contain either of two modified sugars, N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc), and a uronic acid such as glucuronate or iduronate.
Proteoglycans (mucoproteins) are formed of glycosaminoglycans (GAGs) covalently attached to the core proteins. They are found in all connective tissues, extracellular matrix (ECM) and on the surfaces of many cell types. Proteoglycans are remarkable for their diversity (different cores, different numbers of GAGs with various lengths and compositions).5
The specific GAGs of physiological significance are:
Hyaluronic acid: (D-glucuronate + GlcNAc) n
Occurrence: Synovial fluid, extracellular matrix (ECM) of loose connective tissue. Serves as a lubricant and shock absorber.
Hyaluronic acid is unique among the GAGs because it does not contain any sulfate and is not found covalently attached to proteins. It forms non-covalently linked complexes with Proteoglycans in the ECM.
Hyaluronic acid polymers are very large (100–10,000 kDa) and can displace a large volume of water.
Dermatan sulfate: (L-iduronate + GalNAc sulfate) n
Occurrence: Skin, blood vessels, heart valves
Chondroitin sulfate: (D-glucuronate + GalNAc sulfate) n
Occurrence: Cartilage, tendons, ligaments, heart valves and aorta.
It is the most abundant GAG.
Heparin and heparan sulfate: (D-glucuronate sulfate + N-sulfo-D-glucosamine) n
Heparans have less sulfate groups than heparins.
Occurrence:
- Heparin: Component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin (contrary to other GAGs that are extracellular compounds, it is intracellular).
- Heparan sulfate: Basement membranes, component of cell surfaces.
Keratan sulfate: (Gal + GlcNAc sulfate) n
Occurrence: Cornea, bone, cartilage.
Keratan sulfates are often aggregated with Chondroitin sulfates.
Structure of Proteoglycans
The GAGs extend perpendicular from the core protein in a bottlebrush—like structure.
The linkage of GAGs such as (heparan sulfates and Chondroitin sulfates) to the protein core involves a specific trisaccharide linker.
Some forms of keratan sulfates are linked to the protein core through an N-asparaginyl bond.
The protein cores of Proteoglycans are rich in Ser and Thr residues which allow multiple GAG attachments.
Role of Proteoglycans and Glycosaminoglycans
They perform numerous vital functions within the body.
Glycosaminoglycan dependent functions can be divided into two classes: the biophysical and the biochemical.
- The biophysical functions depend on the unique properties of GAGs: The ability to fill the space, bind and organize water molecules and repel negatively charged molecules. Because of high viscosity and low compressibility they are ideal for a lubricating fluid in the joints. On the other hand their rigidity provides structural integrity to the cells and allows the cell migration due to providing the passageways between cells.
- The other, more biochemical functions of GAGs are mediated by specific binding of GAGs to other macromolecules, mostly proteins. Proteoglycans participate in cell and tissue development and physiology.
✓ Case Study 3
A 12-year-old child was brought to the medical outpatient department (OPD) to seek medical help as he was having constant dribbling of thick mucous from mouth and was not responding to surroundings. The mother of the child reported that the child stopped making developmental progress at age of 2 years and developed coarse facial features with thick mucosal drainage. Skeletal deformities appeared over the next year, and the child regressed to a vegetative state by age of 10 years.
The attending physician suspected a metabolic disorder from the history and clinical symptoms. A complete urine analysis was done and that revealed the presence of Heparan sulfate and dermatan sulfate.
What is the probable diagnosis of this patient?
CASE DISCUSSION
The child is suffering from Hurler's syndrome. It is a type of Mucopolysaccharidosis. Inborn errors of glycosaminoglycans degradation cause neurodegradation and physical stigmata described by the outmoded term “gargoylism”.
Glycosaminoglycans are long, negatively charged, unbranched, heteropolysaccharide chains, generally composed of a repeating disaccharide unit {acidic sugar-amino sugar} n.
Mucopolysaccharidosis
Glycosaminoglycans are degraded by Lysosomal Hydrolases. A deficiency of one of the Hydrolase results in a Mucopolysaccharidosis. These are hereditary disorders, in which the Glycosaminoglycans accumulate in tissues, causing symptoms such as skeletal and extracellular matrix deformities, and mental retardation.
CASE DETAILS
Hurler's Syndrome
It is a Mucopolysaccharidosis-I (MPS I)
MPS I is divided into three subtypes based on severity of symptoms. All three types result from an absence of, or insufficient levels of, the enzyme α-L-Iduronidase. MPS I H or Hurler's syndrome is the most severe of the MPS I subtypes. The other two types are MPS I S or Scheie syndrome and MPS I H-S or Hurler-Scheie syndrome.
BASIC DEFECT
There is deficiency of α-L-Iduronidase enzyme. This enzyme removes Iduronic acid from the chain and then subsequently, the other residues are removed by the specific enzymes. In its deficiency degradation of dermatan sulfate and heparan sulfate is affected. These GAGs (Gylcosaminoglycans) get accumulated in the tissues producing a variety of symptoms characteristic of this disease. These GAGs are also excreted excessively in urine.
INHERITANCE
This disease is Autosomal recessive in nature.
FREQUENCY
Approximately 1 in 150,000 infants are affected.
CLINICAL MANIFESTATIONS
- The condition is marked by progressive deterioration, hepatosplenomegaly, dwarfism and gargoyle-like faces. There is a progressive mental retardation, with death frequently occurring by the age of 10 years.
- Newborn infants with this defect appear normal at birth, developmental delay is evident by the end of the first year, and patients usually stop developing between ages 2 and 4. This is followed by progressive mental decline and loss of physical skills.
- Language may be limited due to hearing loss and an enlarged tongue.
- Affected children may be large at birth and appear normal but may have inguinal or umbilical hernias.
- Growth in height may be initially faster than normal, then begins to slow before the end of the first year and often ends around age 3.
- Many children develop a short body trunk and a maximum stature of less than 4 feet.
- Distinct facial features (including flat face, depressed nasal bridge, and bulging forehead) become more evident in the second year (Figure 1). By age 2, the ribs have widened and are oar-shaped.
- Carpal tunnel syndrome (or similar compression of nerves elsewhere in the body) and restricted joint movement are common.
- The children slowly develop corneal clouding and a conductive hearing loss is also present.
- The liver, spleen and heart are often enlarged.
- Children may experience noisy breathing and recurring upper respiratory tract and ear infections.
- Feeding may be difficult for some children, and many experience periodic bowel problems.
- Children with Hurler syndrome often die before age 10 from obstructive airway disease, respiratory infections, or cardiac complications.
DIAGNOSIS
Diagnosis often can be made through clinical examination and laboratory tests.
Laboratory Investigations
- Urine test, which shows the excessive excretion of Heparan and dermatan sulfate. Cetyl Trimethyl ammonium bromide test is undertaken to confirm the presence of gylcosaminoglycans in urine.
- Absence of Lysosomal alpha-L-Iduronidase (in cultured fibroblasts).
- Culture of cells from amniotic fluid obtained by amniocentesis for enzyme testing (prenatal testing).
- X-ray of spine and chest.
Prenatal diagnosis using amniocentesis and chorionic villus sampling can verify if a fetus either carries a copy of the defective gene or is affected with the disorder. Genetic counseling can help parents who have a family history of the Mucopolysaccharidosis determine if they are carrying the mutated gene that causes the disorders.
TREATMENT
This disease can be treated by bone marrow transplantation (BMT) and umbilical cord blood transplantation (UCBT) preferably before the age of 18 months. Abnormal physical characteristics, except for those affecting the skeleton and eyes, can be improved, and neurologic degeneration can often be halted. BMT and UCBT are high-risk procedures with high rates of morbidity and mortality. There is no cure for MPS I.
Gene therapy trials are also going on as a permanent cure for this syndrome. Enzyme replacement therapies are currently in use, they have proven useful in reducing non-neurological symptoms and pain.
SCHEIE SYNDROME
This mild form of MPS I is characterized by joint stiffness, aortic valve disease, corneal clouding, and few other somatic features. Onset of significant symptoms is usually after the age of 5 years, with diagnosis made between 10 and 20 years. Patients with Scheie syndrome have normal intelligence and stature but have significant joint and ocular involvement.
AN OVERVIEW OF TYPES OF MUCOPOLYSACCHARIDOSIS
Main Mucopolysaccharidosis
Type | Main diseases | Deficient enzyme | Accumulated products | Symptoms |
---|---|---|---|---|
MPS I | Hurler's syndrome | α-L-Iduronidase | Heparan sulfate Dermatan sulfate |
|
MPS II | Hunter syndrome | Iduronate sulfatase |
|
|
MPS III | Sanfilippo syndrome A Sanfilippo syndrome B Sanfilippo syndrome C Sanfilippo syndrome D | Heparan sulfamidase N-acetylglucosaminidase Acetyl-CoA:alpha-glucosaminide acetyltransferase N-acetylglucosamine 6-sulfatase |
|
|
MPS IV | Morquio syndrome A Morquio syndrome B | Galactose-6-sulfate sulfatase Beta-galactosidase |
|
|
MPS VI | Maroteaux-Lamy syndrome | N-acetylgalactosamine-4-sulfatase |
|
|
MPS VII | Sly syndrome | β-glucuronidase |
|
|
MPS IX | Natowicz syndrome | Hyaluronidase |
|
|
Clinical Note
Hurler's syndrome is marked by progressive deterioration, hepatosplenomegaly, dwarfism and gargoyle-like faces.
Clinical Pearls
- The basic defect in Hurler's syndrome is deficiency of α-L-Iduronidase enzyme.
- Newborn infants with this defect appear normal at birth, developmental delay is evident by the end of the first year, and patients usually stop developing between ages 2 and 4.
- Hurler's syndrome is marked by progressive deterioration, hepatosplenomegaly, dwarfism and gargoyle-like faces.
- Urine test, which shows the excessive excretion of Heparan and dermatan sulfate.
- This disease can be treated by bone marrow transplantation (BMT) and umbilical cord blood transplantation (UCBT).