eChapters

Biological assay or bioassay

• A bioassay is an analytical method to determine the concentration or potency of a substance by its effect on living animals or plants (in vivo), or on living cells or tissues (in vitro).

• Bio-assays are thus a type of experiments with the object of comparing the efficacy of two or more substances, or preparations, like drugs, by using responses produced by them on suitable living organisms.

• The bio assay involves a stimulus applied to a subject and the response of the subject to the stimulus.

• When a stimulus is applied to a subject there may be a change in some characteristics of the subject. Such changes in the subject are known as responses.

• The response may be quantitative as in the case of weight or qualitative as in the case of mortality. The magnitude of response depends upon the dose.

• Normally, two preparations having a common effect are taken for assaying. One of the preparations is of known strength and is called the standard preparation and the other is of unknown strength and is called test preparation.

Introduction

Urinalysis, the examination of normal and abnormal components of urine, is one of the most important aspect of clinical pathology that aids in accurate and timely diagnosis in veterinary practice. Urine is considered as one of the most accessible bio-fluid that can be collected using non- invasive techniques in adequate quantities. It includes the evaluation of colour, odour, turbidity, volume, pH, specific gravity, protein and glucose content, ketone bodies, blood, erythrocytes, leukocytes, epithelial cells, casts, crystal, micro-organisms, etc. Urinalysis gives us valuable information regarding the several important attributes of renal function. For example, specific gravity of urine gives valuable insights over the potential of the Henle’s loop to dilute urine and the distal convoluted tubules to concentrate urine after ruling out the extra-renal factors affecting the urine specific gravity. It is highly useful to evaluate the different types of urogenital diseases and it also provides significant information regarding several systemic disorders. It is comparatively quicker to perform and is relatively a low-cost test that could be easily done in most clinical setups.

Introduction

Energy is the major source required to maintain the structure and function of all living cells. This energy is derived from oxidation of carbohydrates, lipids and proteins that is taken in the form of diet. The energy liberated from the process of oxidation is generally captured in the form of ATP, which is known as energy currency of the living cells. Each gram of carbohydrate and protein gives about 4 Kcal on oxidation, while each gram of fat gives about 9 Kcal. Oxidation means addition of oxygen or removal of hydrogen from a compound or removal of electron from an ion or atom increasing its positive charge. Reduction means removal of oxygen or addition of hydrogen to a compound or addition of electron to an ion or atom decreasing its positive charge. Commonly the oxidation reactions are conjugated with reduction reactions and known as redox reactions. Redox reactions are often associated with release of energy. The redox chain constitutes chain of different compounds of increasing redox potentials between hydrogen and oxygen. The living cells depend on redox reactions for their energy requirements. The reactions start by removal of H2 from the substrate, which is then transferred to different components of redox chain, then finally to oxygen to form water. Components of redox chains have a redox potential higher than hydrogen and lower than oxygen.

Introduction

Biochemistry of blood is extremely complex. It plays vital role in maintaining homeostasis with in the body. Blood is a specialized connective tissue which is a fluid that circulates all through the body in cardio vascular system. Blood consists of 55 per cent of plasma and 45 per cent of cellular components.

Plasma: A straw coloured liquid component of blood. Understanding the chemical basis of plasma is very crucial for diagnosis and treating various conditions. The compositional breakdown of plasma can be put forth as 90-92 % of water that acts as transport medium for the remaining 8-10 % of the solutes. Solutes further can be classified as electrolytes (salts), proteins, Lipids, other nutrients and waste products.

Plasma electrolytes: These are the minerals found in the blood that are electrically charged. Their role is very crucial in lot of physiological activities like

• Maintaining the balance of fluids interior and exterior to the cells.

• Carries electrical signals all through the body

• Helps in contraction of muscles

• Regulates blood pressure

Introduction

Carbohydrates, the aldehyde/ ketone derivatives of the higher polyhydric alcohols, or compounds that yield these derivatives on hydrolysis, constitute of mainly four groups viz. monosaccharides, disaccharides, oligosaccharides and polysaccharides. Energy comes mostly from carbohydrates, and products of their degradation are also used for the synthesis of other substances including fatty acids, cholesterol, amino acids etc They are also components of compound lipids, conjugated proteins, and some of their derivatives can even function as medications (e.g., cardiac glycosides, etc). Disorders such as glycogen storage disorders (GSDs) and galactosemia can be brought on by an inherited lack of certain enzymes in the metabolic pathways of various carbohydrates. Diabetes mellitus is characterized by disruption of the metabolism of glucose.

In ruminants, the end products of digestion are largely volatile fatty acids viz. acetic acid, propionic acid and butyric acid and some monosaccharides. As they pass through the ruminal epithelium, they undergo different degrees of metabolism. The fate of volatile fatty acids are as follows: Acetate as precursor to Acetyl CoA (TCA cycle; fat synthesis); Propionate as precursor to glucose (glycolysis; milk lactose) and Butyrate as precursor to Acetyl CoA (TCA cycle). All the digested and absorbed monosaccharides and volatile fatty acids enter into the liver, where two mechanisms operate such as withdrawal of carbohydrates from blood (e.g. glycogenesis; glycolysis etc) and release of glucose by liver to blood (e.g. glycogenolysis; gluconeogenesis etc). In case of monogastrics, the processes of digestion/ fermentation of carbohydrates especially occur in the small intestine. The fate of absorbed glucose and metabolic conversions in the animal body mainly comprise of storage as glycogen in liver and muscle (small amount); oxidation of glucose for providing energy (via Glycolysis pathway; other alternative pathways such as Hexose Monophosphate Pathway (HMP)/ HMP Shunt and Uronic acid pathway etc) and fat synthesis as well as storage in the form of fat in adipose tissue.

Introduction

Carbohydrates are essential organic biomolecules/macromolecules composed of carbon c., hydrogen (H), and oxygen (O) atoms, typically in a 1:2:1 ratio. They are one of the four major classes of biomolecules, alongside proteins, lipids, and nucleic acids. Carbohydrates play critical roles in energy storage, structural integrity and cell signaling. Carbohydrates with an aldehyde as their functional group are called Aldoses. Those with keto as a functional group are called Ketoses.

Classification of Carbohydrates: They are broadly classified into three major groups:

1. Monosaccharides: The simplest form of carbohydrates, consisting of a single sugar unit. They cannot be hydrolyzed to yield simpler forms of sugar. They can be subdivided into trioses, tetroses, pentoses, hexoses, heptoses and octoses etc., depending upon the number of carbon atoms they possess. Important monosaccharides are:

• Glycerose / Glyceraldehyde (Aldotriose): Glyceraldehyde 6-phosphate is an intermediate in Glycolysis.

• Ribose (Aldopentose): found in Nucleic acids.

• Glucose (Aldohexose): The primary energy source for cells, known as blood sugar.

• Fructose (Ketohexose): Found in fruits (fruit sugar), a major component of sucrose.

• Galactose (Aldohexose): Known as brain sugar. It is a part of lactose sugar.

Introduction

Energy metabolism is the complex process by which living organisms convert nutrients into energy for survival, growth, and reproduction. It involves a series of interconnected biochemical reactions that occur within cells.

Key Components of Energy Metabolism

• Catabolism: The breakdown of complex molecules (carbohydrates, proteins, and fats) into simpler ones, releasing energy.

• Anabolism: The synthesis of complex molecules from simpler ones, requiring energy input.

• ATP: The primary energy currency of cells, used to power various cellular processes.

• Enzymes: Catalysts that facilitate metabolic reactions.

Introduction

All enzymes are proteins, except a tiny subset of catalytic RNA molecules. Most of the classical metabolic enzymes are named by adding the suffix -ase to the name of their substrates or to a descriptive term for the reactions they catalyze. For example, urease has urea as a substrate. Alcohol dehydrogenase catalyzes the removal of hydrogen from alcohols (i.e., the oxidation of alcohols). A committee of the International Union of Biochemistry and Molecular Biology (IUBMB maintains a classification scheme that categorizes enzymes according to the general class of organic chemical reaction that is catalyzed The six classes are Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and Ligases.

The integrity of their natural protein structure is necessary for their catalytic activity. Catalytic activity is typically lost when an enzyme denatures or dissociates into its constituent subunits. An enzyme’s catalytic activity is always eliminated when it is disassembled into its constituent amino acids. Thus, the catalytic activity of protein enzymes depends on their primary, secondary, tertiary, and quaternary structures. Aside from their amino acid residues, certain enzymes don’t need any other chemical groups to function. Others need a cofactor, which is an extra chemical element. Cofactors can be complex organic or metalloorganic molecules called coenzymes, or they can be one or more inorganic ions like Fe2+, Mg2+, Mn2+, or Zn2+.

Introduction

Most glands of the body deliver their secretions by means of ducts. These are called exocrine glands. There are few glands that produce chemical substance that they directly secrete into the blood stream for transmission to various target tissues. These are ductless or endocrine glands. The secretions of endocrine glands are called as hormones. Therefore, it is a chemical substance which is produced in one part of the body, enters the circulation and is carried to distant target organs and tissues to modify their structures and functions. Hormones are stimulating substances and act as body catalysts. The hormones catalyze and control diverse metabolic processes. Despite their varying actions and different specificity’s depending on the target organ, the hormones have several characteristics in common with enzymes. They act as body catalysts resembling enzymes in some aspect. They are required only in small quantities. They are not used up during the reaction. They differ from enzymes because they are produced in an organ other than that in which they ultimately perform their action. The major hormone secreting glands are as follows— Pituitary, Thyroid, Parathyroid, Adrenal, Pancreas, Ovaries and Testes. The hormones are classified into following main classes according to the chemical structures:

Introduction

Lipids (dietary or body reserve) serves as the major source of energy for the body at the time of carbohydrate depletion. Triacyl glycerol (TG) forms the major part of lipids alongwith phospholipids, cholesterol and fat-soluble vitamins. The different forms of lipids are utilised by the body for generation of energy in the form of ATP. In addition, it serves as the source of essential fattyacids and vitamins for different metabolic purpose.

Dietary triacylglycerols (TG) are digested by lipase group of enzymes (lingual, gastric, pancreatic and intestinal) secreted by the digestive tract into free fatty acids and 2-monoacylglycerols. The 2-monoacylglycerols are either absorbed as such or hydrolysed into glycerol and fatty acids which are then absorbed independently. The glycerol and fatty acids are distributed to different tissues through circulation and utilised in the following ways

i. Storage in fat cells of adipose tissue in the form of triacylglycerols.

ii. Oxidised in peripheral tissues for production of energy.

iii. converted to glucose in hepatic cells in the process of gluconeogenesis.

iv. Synthesis of other derived compounds like steroids and eicosanoids in gonadal and other tissues

v. Synthesis of phospholipids and cholesteryl esters.

Introduction

Lipids may be regarded as organic substances relatively insoluble in water, soluble in organic solvents (chloroform, ether, benzene, ethanol and other non-polar solvents, etc.), actually or potentially related to fatty acids and utilized by living cells. Lipids are a diverse group of hydrophobic or amphipathic biomolecules that play crucial roles in energy storage, cell membrane structure, and signaling. Unlike carbohydrates and proteins, lipids are not defined by a specific polymer structure but rather by their solubility in non-polar solvents. Broadly lipids are classified based on their function as ‘Storage lipids’ and ‘Structural lipids’. Some of them are co-factors, hormones, electron carriers, light-absorbing pigments and hydrophobic anchors of proteins.

Fatty Acids: Fatty acids are straight aliphatic chains with a methyl group at one end and a carboxyl group (-COOH) at the other end. They are the simplest form of lipids.

• Saturated Fatty Acids have no double bonds between carbon atoms, leading to straight chains (e.g., palmitic acid).

• Unsaturated Fatty Acids contain one or more double bonds, causing kinks in the chain (e.g., oleic acid). Fatty acids containing more than one double bond (polyunsaturated fatty acids/PUFA) are found in relatively minor amounts.

Introduction

According to the body needs, minerals may be divided into 2 groups like macrominerals and microminerals. Macrominerals are required in amounts greater than 100 mg/day. They include 6 elements like calcium, phosphorus, magnesium, sodium, potassium and chloride. Whereas microminerals are also known as trace elements and are required in amounts less than 100 mg/day. They include 10 elements like chromium, cobalt, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc and silicon. Some minerals are always required since small amounts of inorganic salts are always excreted Fruits, vegetables and cereals are the chief sources of the mineral elements in the diet. Milk products supply the majority of the calcium and phosphorus in the diet.

Milk and milk products are the richest source of calcium along with beans, leafy vegetables and egg yolk. It is absorbed from upper small intestine. Absorption of calcium is active process and requires calcium binding protein present in the intestinal mucosal cells. The absorption is regulated by vitamin D, parathyroid hormone, high protein diet, pH, high dietary lactate and citrate are the factors that promotes calcium absorption. Whereas high dietary phosphate, alkalinity and impaired fat absorption are the factors that inhibits calcium absorption. Of the total body calcium, 99 % present in bones and teeth in the form of hydroxyapatite and 1 % is present in body fluids and other tissues. Calcium in bones acts as a reservoir, which helps to stabilize calcium ions in plasma and extracellular f luid Parathyroid hormone and active vitamin D stimulate osteoblasts while estrogen hormone inhibits osteoclasts.

Introduction

A pentose sugar, one or more phosphate groups, and a nitrogenous base (purine or pyrimidine) make up a nucleotide. Phosphodiester bonds, which connect the 5’-hydroxyl group of one pentose to the 3’-hydroxyl group of the next, bind nucleic acids together as polymers of nucleotides. RNA and DNA are the two different forms of nucleic acids. RNA nucleotides contain ribose, while uracil and cytosine are typical pyrimidine bases. Thymine and cytosine are common pyrimidine bases, and DNA contains 2’-deoxyribose in its nucleotides. In both RNA and DNA, adenine and guanine are the main purines. There is ample evidence to suggest that genetic information is encoded in DNA The Avery MacLeod-McCarty experiment, in particular, demonstrated how DNA obtained from one bacterial strain may infiltrate and change the cells of another strain, giving it some of the genes from the donor. The Hershey-Chase experiment demonstrated that a bacterial virus’s DNA contains the genetic instructions needed for the virus to replicate in a host cell, but not its protein coat. Watson and Crick proposed that native DNA is made up of two antiparallel chains arranged in a right-handed double helical configuration based on a compilation of numerous published data points. A-T and G-C, two complementary base pairs, are created by hydrogen bonds inside the helix. 10.5 base pairs are stacked per turn, perpendicular to the double helix’s long axis, at a distance of 3.4 Å. There are various structural configurations for DNA A- and Z-DNA are two variants of the Watson-Crick form or B-DNA The DNA molecule bends as a result of certain sequence-dependent structural differences.

Introduction

Protein metabolism is a complex and multifaceted process that plays a crucial role in the overall health and function of living organisms. At the most fundamental level, protein metabolism involves the synthesis, breakdown, and utilization of proteins, which are essential macromolecules that serve a wide range of structural, functional, and regulatory roles within the body.

The process of protein synthesis, or translation, begins with the transcription of genetic information stored in DNA into messenger RNA (mRNA) molecules. These mRNA molecules then serve as templates for the assembly of specific sequences of amino acids into polypeptide chains, which ultimately fold into the three-dimensional structures of functional proteins. This highly regulated process is essential for the production of the diverse array of proteins required for cellular processes, tissue repair, immune function, and numerous other physiological activities. In contrast, protein degradation, or catabolism, refers to the breakdown of proteins into their constituent amino acids. This process is mediated by various proteolytic enzymes, such as endopeptidases, exopeptidases, and dipeptidases, which cleave the peptide bonds that hold the amino acid residues together Protein degradation serves important functions, such as the recycling of amino acids for energy production or the synthesis of new proteins, the removal of damaged or misfolded proteins, and the regulation of cellular signaling pathways.

Introduction

Proteins are complex biomolecules composed of amino acids, playing crucial roles in virtually all biological processes. They serve as enzymes, hormones, structural components, signaling molecules etc.

Structure/ Levels of Organization of Proteins

Generally, Proteins are polymers of L-alpha amino acids, linked by peptide bonds. The sequence and structure of these amino acids determine the protein’s function. Protein structure is hierarchically organized into four levels:

1. Primary Structure

• The linear sequence of amino acids in a polypeptide chain, determined by the genetic code.

2. Secondary Structure

• Local folding of the polypeptide chain into structures such as α-helices and β-pleated sheets, stabilized by hydrogen bonds.

Introduction

Human and animal body comprises of various tissues that are the functional unit of organ functioning and coordination. The tissues are further consisting of well differentiated cells that makes the tissue to behave in a special way for a particular purpose. The animal tissues are divided into epithelial, connective, muscular and nervous tissues. Epithelial tissues form the protective covering and inner lining of the body and organs. Connective tissues develop from the mesodermal cells of the embryo. they support and bind other tissues in the body. The muscular tissue develops from the mesoderm of the embryo and meant for movement and locomotion. Nervous tissue makes up the peripheral and the central nervous system and is responsible for initiation and transmission the nerve impulse. The tissues are biochemically composed of water, lipid, protein, ash, and carbohydrate along with other mineral and vitamins that are needed for its basic functioning. The extracellular matrix (ECM) forms a major component of connective tissue that forms layer and surrounds the cells or tissues meant for protection, signalling and communication. The protein component of ECM basically includes collagen, elastin, keratins, fibrillin, fibronectin, laminin and proteoglycans. Collagen is the major protein component of connective tissue that provides tensile strength, shape and fixes the organ in its position. Collagen is not homogenous rather consists of around 19 different types of proteins. There are around 30 variants of protein found in collagen. collagens type I and type II are respectively found in skin and bone. Collagen is triple helical polymer of highly elongated fibrils consisting of around 1000 amino acids majority of which consists of glycine along with proline and hydroxyproline. The forces like hydrogen bonds, covalent bonds, electrostatic, hydrophobic, and van der Waals are responsible for stabilizing the structure of collagen.

Introduction

Vitamins are the organic derived accessory food factors apart from carbohydrate, lipid and proteins meant for specific biological functions, optimum growth and health of an organism. These factors are are required in minute amount in the diet for proper functioning of biological enzymes. The term accessory factors were given by Hopkins to the essential nutrients present in the natural foods. Funk (1913) coined the term vitamine to the active principal present in rice polishings. Vitamins are classified into two categories like fat soluble A and water-soluble b Vitamins A, D, E, K are fat soluble whereas Vitamins B and C are water soluble. The B group of vitamins include vitamin B1, B2, B6, biotin folic acid, B12 etcall the fat-soluble vitamins are isoprenoid in nature consisting of repeating isoprenoid units. And meant for diversified functions. The water-soluble vitamins are heterogenous group of compounds generally not stored in the body and readily excreted in urine. These are mostly act as coenzymes for the vital biological enzymes deficiency of which reflects in the form of various deceases.

Vitamin A, include retinoid group of compounds like retinol, retinal and retinoic acid Retinol is a primary alcohol containing β-ionone ring with isoprenoid units, double bonds and hydroxyl group. Naturally it exists as ester of long chain fatty acids. Retinal is the aldehyde form whereas Retinoic acid is the acid form. Carotene is provitamin A normally found in plant origin foods that in the intestine gives rise to two moles of retinal by enzyme carotene 15-15-dioxygenase. Diet contains retinol esters and p-carotene. Retinol esters are hydrolyzed into fatty acids and retinol that absorbed into intestinal mucosal cells. p-carotene Is absorbed and converted into retinal by p-carotene dioxygenase. Retinal then converted into retinol. In the intestinal mucosal cells, retinol re-esterifies with fatty acid to form retinol ester.

Biodiversity, coined by W. G. Rosen in 1985, is the variety of life on Earth. It is the occurrence of different kinds of organisms and the complete range of varieties adapted to different climates, environments and areas being constituents of food chains and food web of biotic interrelationship. Biodiversity refers to totality of genes species and ecosystems of a region. Biodiversity differs from place to place. Biodiversity is the biological wealth of world.

There is continuous depletion of biodiversity due to increasing population resources consumption, urbanization and pollution. The basic reason for concern is that biodiversity is being lost even before it attains its size. Loss of biodiversity would check evolutionary capability of biota to cope up with environmental loss.

14.1 Ethics in Scientific Research and Innovation
Introduction
Scientific research and innovation in biotechnology, particularly in the realms of genetic engineering, genomics, and transgenic organisms, bring immense promise for advancing agriculture, health, and environmental sustainability. However, this progress must be balanced with strong ethical foundations, as the misuse or careless application of biotechnological innovations can lead to irreversible harm to humans, society, and the biosphere. The term bioethics encapsulates the principles of morality, justice, and responsibility that should govern all scientific inquiry and technological development.
Ethics in scientific research and innovation is not merely an adjunct to regulatory compliance—it is a core component of scientific integrity and social responsibility. It ensures that biotechnological advances are developed in ways that respect human dignity, safeguard biodiversity, and reflect societal values.
1. Fundamental Ethical Principles in Scientific Research
Ethical decision-making in science is guided by several foundational principles, adapted from biomedical and research ethics. These include:

Bioinformatics is a branch of biological science which deals with the study of methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. It generates new knowledge that is useful in such fields as drug design and development of new software tools to create that knowledge. Bioinformatics also deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, structural biology, software engineering, data mining, image processing, modeling and simulation, discrete mathematics, control and system theory, circuit theory, and statistics.