Factors affecting biotransformation of drugs.

 

• Biotransformation of drugs is defined as the chemical conversion of one form to another. The term is used synonymously with metabolism,

• The therapeutic efficacy, toxicity and biological half-life of a drug greatly depend upon its metabolic rate. 

• A number of factors may influence the rate of drug metabolism. They are as follows,

1. Physicochemical properties of the drug

2. Chemical factors:

• Induction of drug metabolizing enzymes

• Inhibition of drug metabolizing enzymes

• Environmental chemicals

3. Biological factors:

• Species differences

• Strain differences

• Sex differences

• Age

• Diet

4. Altered physiological factors:

• Pregnancy

• Hormonal imbalance

• Disease states

5. Temporal factors:

• Circadian rhythm

• Circannual rhythm.

1. Physicochemical properties of the drug:

• Just as the absorption and distribution of a drug are influenced by its physicochemical properties, so is its interaction with the drug metabolizing enzymes. 

• Molecular size and shape, pKa, acidity/basicity, lipophilicity and steric and electronic characteristics of a drug influence its interaction with the active sites of enzymes and the biotransformation processes to which it is subjected. 

• Stereochemical nature of the drug also influences its metabolism. 

2. Chemical factors:

1. Induction of drug metabolizing enzymes

2. Inhibition of drug metabolizing enzymes

3. Environmental chemicals

1. Induction of drug metabolizing enzymes:

• Certain drugs stimulate the enzyme system causing increased metabolism, the phenomenon is called enzyme induction and the enzyme is called enzyme inducer.

• e.g. 

• Consequences of enzyme induction includes –

• Decrease in pharmacological activity of drugs.

• Increased activity where the metabolites are active, and

• Altered physiologic status due to enhanced metabolism of endogenous compounds such as sex hormones.

2. Inhibition of drug metabolizing enzymes:

• Certain drugs inhibit the enzyme system causing decreased metabolism, the phenomenon is called enzyme inhibition and the enzyme is called enzyme inhibitor.

• e.g. 

• The process of inhibition may be direct or indirect.

• 1. Direct Inhibition: may result from interaction at the enzymic site, the net outcome being a change in enzyme activity.

• Direct enzyme inhibition can occur by one of the 3 mechanisms –

• a. Competitive Inhibition: 

• Occurs when structurally similar compounds compete for the same site on an enzyme. 

• Such an inhibition due to substrate competition is reversible and can be overcome by high concentration of one of the substrates, 

• e.g. PABA and Sulfa drugs.

• b. Non-competitive Inhibition: 

• Occurs when a structurally unrelated agent interacts with the enzyme and prevents the metabolism of drugs. 

• Since the interaction is not structure-specific, metals like lead, mercury and arsenic and organophosphorus insecticides inhibit the enzymes non-competitively. 

• e.g. Isoniazid inhibits the metabolism of phenytoin.

• c. Product Inhibition: 

• Occurs when the metabolic product competes with the substrate for the same enzyme. The phenomenon is also called autoinhibition.

• Certain specific inhibitors such as xanthine oxidase inhibitors (e.g. allopurinol) and MAO inhibitors (e.g. phenelzine) also inhibit the enzyme activity directly. 

• Direct enzyme inhibition is usually rapid; a single dose of inhibitor may be sufficient to cause enzyme inhibition.

• 2. Indirect Inhibition: is caused by one of the two mechanisms –

• a. Repression: 

• It is defined as the decrease in enzyme content. 

• It may be due to a fall in the rate of enzyme synthesis as affected by ethionine, puromycin and actinomycin D or because of rise in the rate of enzyme degradation such as by carbon tetrachloride, disulfiram, etc.

• b. Altered Physiology: due to nutritional deficiency or hormonal imbalance.

• Enzyme inhibition is more important clinically than enzyme induction, especially for drugs with narrow therapeutic index, e.g. anticoagulants, antiepileptics, hypoglycemics, since it results in prolonged pharmacological action with increased possibility of toxic effects.

3. Environmental chemicals:

• Several environmental agents influence the drug metabolizing ability of enzymes.

• e.g.

• Halogenated pesticides such as DDT and polycyclic aromatic hydrocarbons contained in cigarette smoke have enzyme induction effects.

• Organophosphate insecticides and heavy metals such as mercury, tin, nickel, cobalt and arsenic inhibit drug metabolizing ability of enzymes.

• Other environmental factors that may influence drug metabolism are temperature, altitude, pressure, atmosphere, etc.

3) Biological factors:

1. Species differences

2. Strain differences

3. Sex differences

4. Age

5. Diet.

1. Species differences:

• Species differences have been observed in both phase I and phase II reactions. 

• An example of this is the metabolism of amphetamine and ephedrine. 

• In men and rabbits, these drugs are predominantly metabolized by oxidative deamination whereas in rats the aromatic oxidation is the major route. 

2. Strain differences:

• Enzymes influencing metabolic reactions are under the genetic control. 

• Just as the differences in drug metabolizing ability between different species are attributed to genetics, so also are the differences observed between strains of the same animal species.

• In identical twins (monozygotic), very little or no difference in the metabolism of phenylbutazone, dicoumarol and antipyrine was detected but large variations were apparent in fraternal twins (dizygotic; twins developed from two different eggs) for the same drugs.

• Some humans metabolize Isoniazid faster; they are called fast acetylators and while those who show slower metabolism are called slow acetylators.

3. Age:

• Differences in the drug metabolic rate in different age groups are mainly due to variations in the enzyme content, enzyme activity and haemodynamics.

• In neonates (upto 2 months), the microsomal enzyme system is not fully developed and many drugs are biotransformed slowly. 

• chloramphenicol leads to cyanosis or Gray baby syndrome in newborns.

• sulfonamides cause renal toxicity and paracetamol causes hepatotoxicity.

• Infants (between 2 months and one year) show almost a similar profile as neonates in metabolizing drugs with improvement in the capacity as age advances and enzyme activity increases.

• Children (between one year and 12 years) and older infants metabolize several drugs much more rapidly than adults As a result, they require large mg/Kg doses in comparison to adults; for example, theophylline.

• In very elderly persons, the liver size is reduced, the microsomal enzyme activity is decreased and hepatic blood flow also declines as a result of reduced cardiac output all of which contribute to decreased metabolism of drugs. 

4. Diet:

• The enzyme content and activity is modified by a number of dietary components.         

• Low protein diet decreases and high protein diet increases the drug metabolizing ability. 

• This is because the enzyme synthesis is promoted by protein diet which also raises the level of amino acids for conjugation with drugs.

• Fat free diet depresses cytochrome P-450 levels since phospholipids, which are important components of microsomes, become deficient.

• Dietary deficiency of vitamins (e.g. vitamin A, B2, B3, C and E) and minerals such as Fe, Ca, Mg, Cu and Zn retard the metabolic activity of enzymes.

• Grapefruit inhibits metabolism of many drugs and improves their oral availability.

• Malnutrition in women results in enhanced metabolism of sex hormones.

• Alcohol ingestion results in a short-term decrease followed by an increase in the enzyme activity.

5. Diseased States:

• Liver is the major site for metabolism of most drugs, all disease conditions associated with it result in enhanced half-lives of almost all drugs. 

• Thus, a reduction in hepatic drug metabolizing ability is apparent in conditions such as hepatic carcinoma, hepatitis, cirrhosis, obstructive jaundice, etc. 

• Biotransformations such as glycine conjugation of salicylates, oxidation of vitamin D and hydrolysis of procaine which occur in the kidney, are impaired in renal diseases. 

• Congestive cardiac failure and myocardial infarction which result in a decrease in the blood flow to the liver, impair metabolism of drugs having high hepatic extraction ratio e.g. propranolol and lidocaine. 

• In diabetes, glucuronidation is reduced due to decreased availability of UDPGA.

4) Altered physiological factors:

1. Pregnancy

2. Hormonal imbalance

3. Disease states

1. Pregnancy:

• Studies in animals have shown that the maternal drug metabolizing ability (of both phase I and phase II reactions) is reduced during the later stages of pregnancy. 

• This was suggested as due to high levels of steroid hormones in circulation during pregnancy. 

2. Hormonal imbalance:

• Higher levels of one hormone may inhibit the activity of few enzymes while inducing that of others. 

• e.g. Stress related changes in ACTH levels also influence drug biotransformation.

5) Temporal factors:

1. Circadian rhythm

1. Circadian rhythm:

• Variations in the enzyme activity with light cycle are called as circadian rhythm in drug metabolism. 

• It has been observed that the enzyme activity is maximum during early morning (6 to 9 a.m.) and minimum in late afternoon (2 to 5 p.m.) which was suggested to correspond with the high and low serum levels of corticosterone. 

• The study of variations in drug response as influenced by time is called chronopharmacology. 

• Time dependent change in drug kinetics is known as chronokinetics. 

Commonly Asked Question.

1. Discuss different factors affecting drug biotransformation.

Introduction to Proteins.

 

Definition:

These are high molecular weight biomolecules made up of chains of amino acids linked together with “peptide bonds”, found in all living organisms.

Biological Functions of Proteins:

1. Proteins, which are composed of amino acids, serve in many roles in the body (e.g., as enzymes, structural components, hormones, and antibodies).

2. They are structural components of hair and nail, collagen of bone etc e.g. keratin.

3. Through Proteins genetic information is expressed.

4. Gaseous transportation e.g. Hemoglobin in RBCs of Blood.

5. Homeostatic control of the volume of the circulating blood and that of the interstitial fluids through the plasma proteins.

6. Blood clotting through thrombin, fibrinogen and other protein factors.

7. Defense against infections through protein antibodies.

8. Hereditary transmission by nucleoproteins of the cell nucleus.

9. Ovalbumine, glutelin etc. are storage proteins.

10. Myosin acts as a contractile protein important for muscle contraction.

Classification of Proteins:

• Depending on their chemical nature the proteins are classified as follows,

1. Simple Proteins:

• They are composed of only amino acid residue. 

• On hydrolysis these proteins yield only constituent amino acids.

•  It is further divided into:

• Fibrous protein: Keratin, Elastin, Collagen

• Globular protein: Albumin, Globulin, Glutelin, Histones.;

2. Conjugated proteins:

• They are combined with non-protein moiety. Eg. Nucleoprotein, Phosphoprotein, Lipoprotein, Metalloprotein etc.

• The non protein moiety is called the “Prosthetic group”.

• Nucleoprotein: Combination of proteins with RNA or DNA.

• Chromoproteins: Combination of proteins with colored components e.g. Globin with Heme (Red Color) forms Hemoglobin.

• Glycoproteins: Combination of proteins with carbohydrates.

• Lipoproteins:  Combination of proteins with lipids.

• Metalloproteins:  Combination of proteins with metals.

3. Derived proteins:

• They are derivatives or degraded products of simple and conjugated proteins. They may be :

• Primary derived protein: Proteans, Metaproteins, Coagulated proteins

• Secondary derived proteins: Proteosans or albunoses, peptones, peptides.

Structure of Proteins:

• The linear sequence of amino acids in a polypeptide chain determines the three-dimensional configuration of a protein, and the structure of a protein determines its function.

• The structure of proteins can be divided into four levels of organization:

• Primary Structure.

• Secondary Structure.

• Tertiary Structure.

• Quaternary Structure.

• Primary Structure:

• The primary structure of a protein consists of the amino acid sequence along the polypeptide chain.

• Amino acids are joined by peptide bonds.

• The charges on a polypeptide chain are due only to the N-terminal amino group, the C-terminal carboxyl group, and the side chains on amino acids.

• The primary structure determines the further levels of organization of protein molecules.

• Secondary Structure:

• In a protein molecule, the polypeptide is present in different geometric configurations.

• These arrangements are due to hydrogen bonding between carboxyl groups and amino groups of the peptide bonds.

• The atoms of the side chains are not involved.

• This is called the secondary structure of the protein.

• It is of two types,

• Alpha-helix

• Beta-helix

• Alpha-helix:

• The α-helix is a right-handed coiled strand.

• The side-chain substituents of the amino acid groups in an α-helix extend to the outside.

• Hydrogen bonds form between the oxygen of the C=O of each peptide bond in the strand and the hydrogen of the N-H group of the peptide bond four amino acids below it in the helix.

• The side-chain substituents of the amino acids fit in beside the N-H groups.

• The sheet conformation consists of pairs of strands lying side-by-side.

• The two strands can be either parallel or antiparallel.

• Hair protein Keratin is made up of Alpha helix.

• Beta Helix:

• In this arrangement, the polypeptide chains are stretched out beside one another and then bonded by intermolecular H-bonds.

•  In this structure, all peptide chains are stretched out to nearly maximum extension and then laid side by side which is held together by intermolecular hydrogen bonds. 

• The structure resembles the pleated folds and therefore is known as β – pleated sheet.

• Tertiary Structure:

• This structure arises from further folding of the secondary structure of the protein, due to sidechain interactions.

• H-bonds, electrostatic forces, disulphide linkages, and Vander Waals forces stabilize this structure.

• The tertiary structure of proteins represents overall folding of the polypeptide chains, further folding of the secondary structure.

• It gives rise to two major molecular shapes called fibrous and globular.

• The main forces which stabilize the secondary and tertiary structures of proteins are hydrogen bonds, disulphide linkages, van der Waals and electrostatic forces of attraction.

• Quaternary Structure:

• Quaternary structure refers to the interaction of one or more subunits to form a functional protein, using the same forces that stabilize the tertiary structure.

• It is the spatial arrangement of subunits in a protein that consists of more than one polypeptide chain.

• e.g. Hemoglobin (𝝰2𝞫2).

Peptides and Polypeptides:

• A peptide is a condensation product of two or more amino acids.

• A dipeptide is a condensation product of two amino acids.

• A tripeptide is a condensation product of three amino acids and so on.

• The term peptide is usually used for a product containing 2-20 amino acids e.g. Oxytocin, Vasopressin.

• The term polypeptide is usually used for a product containing 20-50 amino acids, e.g. Insuline.

• The products containing more than 50 amino acids are called “Proteins”.

Qualitative Tests of Proteins:

1. Heat Test:

• When a protein solution is heated it causes loss of its protein structure which results in denaturation losing its biological activity.

2. Test with Trichloroacetic Acid (TCA):

• TCA causes denaturation of protein and is used for deproteinization.

3. Biuret Test:

• Also known as Piotrowski's test, is a chemical test used for detecting the presence of peptide bonds.

• In the presence of peptides, a copper(II) ion forms violet colored complexes in an alkaline solution.

• Biuret reagent contains copper sulphate in an alkaline medium, it reacts with protein solution forming violet color.

4. Xanthoprotic Test:

• The xanthoproteic test is performed for the detection of aromatic amino acids (tyrosine, tryptophan, and phenylalanine) in a protein solution. 

• The nitration of the aromatic amino acid chain occurs due to reaction with nitric acid, giving the solution yellow coloration.

5. Millon’s Test:

• Millon’s test is an analytical test used for the detection of the amino acid tyrosine.;

• Millon’s test is based on the principle of nitrification of the phenol group in tyrosine, which then forms complexes with heavy metals like mercury. 

• The reagent used for the test is called Millon’s reagent, and it consists of mercuric nitrate and mercurous nitrate that is dissolved in concentrated nitric acid.

6. Precipitation Test:

• Proteins get precipitated by various agents like salts, heavy metals, tannins, organic solvents etc.

Biochemical importance of Proteins:

• Proteins are one of the important components of the diet.

• They are required for normal growth and functioning of the body.

• Nutritionally they are classified in two categories as,

• Complete Proteins.

• Incomplete Proteins.

• Complete Proteins:

• These proteins contain all essential amino acids in required quantities.

• e.g. Milk proteins, Egg Proteins.

• Incomplete Proteins:

• These proteins do not contain all essential amino acids in required quantities.

• e.g. Gelatin.

• Proteins are mainly used for making new components and very rarely are used as a source of energy.

Deficiency Disorders of Proteins:

• Proteins are required for several vital processes in the body, their deficiency leads to deficiency disorders which may be caused due to low dietary intake or malfunctioning in the body like malabsorption and faulty conversions.

• Following are two main deficiency disorders of proteins.

• Marasmus.

• Kwashiorkor.

• Marasmus:

• Marasmus is a severe form of protein-energy malnutrition that results when a person does not consume enough protein and carbohydrates.

• Without these vital nutrients, energy levels become dangerously low and vital functions begin to stop.

• Causes:

• Not having enough nutrition or having too little food: Mainly happens in poor people.

• Symptoms:

• Failure to grow, known as stunted growth.

• Wasting, or a loss of body tissue and fat.

• Bones become visible under their skin.

• persistent dizziness

• lack of energy

• dry skin

• brittle hair.

• Treatment:

• Diet rich in calories, proteins and other nutritional factors is the best treatment and a way to avoid Marasmus.

• Kwashiorkor:

• Kwashiorkor is a form of severe protein malnutrition characterized by edema and an enlarged liver.

• It is caused by sufficient calorie intake, but with insufficient protein consumption, which distinguishes it from marasmus.

• Kwashiorkor cases occur primarily in areas of poor food supply.

• Causes:

• Not having enough nutrition or having too little food: Mainly happens in poor people.

• Premature termination of breastfeeding.

• Over dilution of cow milk.

• Symptoms:

• Pitting edema: Swelling of ankles and feets.

• Distended abdomen, 

• An enlarged liver, 

• Thinning of hair,

•  Loss of teeth, 

• Skin or hair depigmentation, 

• Dermatitis.

• Anorexia (Loss of appetite)

• Irritability.

• Treatment:

• Diet rich in proteins and other nutritional factors is the best treatment and a way to avoid Kwashiorkor

Commonly Asked Questions.

1. Define and classify proteins.

2. Give qualitative analysis tests for proteins.

3. Give the biological importance of proteins.

4. What are peptides, polypeptides and proteins?

5. What is a peptide bond?

6. Write in detail different structures of proteins.

7. Discuss protein deficiency disorders.