eChapters

Introduction

Colorimetry is a scientific technique widely used across various fields to quantify the concentration of substances based on their color intensity. Colorimetry is a branch of analytical chemistry that focuses on measuring the absorbance or transmission of light by colored solutions. It utilizes the principles of optics and spectroscopy to determine the concentration of a substance in a solution based on its color intensity. This technique relies on the Beer-Lambert law, which states that the absorbance of light by a solution is directly proportional to its concentration and path length.

Principles of Colorimetry

The fundamental principle of colorimetry lies in the interaction of light with matter. When light passes through or is reflected off a substance, certain wavelengths are absorbed, while others are transmitted or reflected. The color we perceive is due to the wavelengths of light that are reflected back to our eyes. In colorimetry, a photometric measurement is made using a colorimeter or spectrophotometer, which quantifies the intensity of light absorbed or transmitted by a sample.

Introduction

Biochemistry is branch of life science which deals with the study of chemical reactions occurring in the living cells and organisms. The term biochemistry was first introduced by German chemist Carl Neuberg in 1903. Since biochemistry is associated with the biological reactions and energy changes associated with these reactions or transformations within the living cell, biochemistry may thus be treated as a discipline in which biological phenomenon are analysed in terms of chemistry. The branch of biochemistry for the same reason has been variously named as “Biological Chemistry” or “Chemical Biology”. Modern biochemistry has two branches descriptive and dynamic biochemistry. Descriptive biochemistry deals with qualitative and quantitative characterization of the various chemical components of cell and the dynamic biochemistry deals with the elucidation of the nature and the mechanism of reactions involving these chemical cell components.

Qualitative analysis of biological sample deals with the examination of the organic or inorganic cell associated components in order to determine its composition. It denotes the presence of various biomolecules, elements or groups of elements that are present in the sampling population. It is possible to determine whether a particular component is present or absent in a sample by performing a qualitative analysis of the sample. Qualitative analysis is the identification of elements, species and/or compounds present in a sample.

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

Biological fluids refer to any liquid substances found within living organisms that play essential roles in physiological functions. These fluids can include blood, urine, cerebrospinal fluid, saliva, digestive juices, rumen fluid, peritoneal fluid, pleural fluid, pericardial fluid, synovial fluid, amniotic fluid, milk etc.

Collection of biological fluids refers to the process of gathering various types of bodily f luids from animals for scientific study or medical purposes. This collection often involves techniques like blood draws, urine collection, cerebrospinal fluid sampling, saliva collection, etc., depending on the specific fluid and the research or diagnostic needs. These f luids are valuable for understanding the health, metabolism, genetics, and other aspects of animal biology. Collecting and analyzing these biological fluids is crucial for understanding health and disease processes, diagnosing illnesses, monitoring treatment effectiveness, and advancing medical and scientific research.

Introduction

Immunology deals with the body defence system consisting of various cells and biomolecules that prevents entry of foreign infectious and toxic agents by different mechanisms. The mammalian immune system operates in two ways like innate or non specific immunity and adaptive or acquired or specific immunity. Innate part constitutes the physical (skin, hair, tears and mucous secretions) containing enzymes that prevents primarily entry of pathogen. It is the first line of defence action, very fast but not adequate. Adaptive form constitutes defensive cells and secreted immunoglobulins. It takes time to develop effectively, especially when the body sees the organism for the first time. Immunoglobulins (Ig) or antibodies are a group of proteins produced from B lymphocytes and plasma cells in response to the presence of any foreign substances. They are 5 basic types like M, A, G, E and D each with specific function. The system can produce antibody molecules, directed to about 1 million different kinds of antigens. All immunoglobulins have a similar basic structure.

Introduction

Diagnostic biochemistry, is a branch of laboratory medicine that focuses on the analysis of bodily fluids to diagnose and monitor diseases. This field plays a crucial role in understanding the biochemical mechanisms underlying various conditions, enabling accurate diagnosis, prognosis, and management. The biological samples for diagnosis include blood, urine, cerebrospinal fluid, pleural fluid and tissues.

Factors Affecting Serum Enzyme Activity or Protein Level

1. Organ Mass and Enzyme Tissue Concentration:

• Higher enzyme concentrations in organs can lead to greater increases in serum enzyme activity during disease. For instance, the liver’s large mass contributes to potential increases in serum ALT activity.

2. Cell Location

• The proximity of cellular enzymes to the blood, urine, or other fluids determines the fluid in which an enzyme’s activity will increase upon cell injury. For example, renal tubular gamma-glutamyltransferase (GGT) is released into urine, not blood, following injury.

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

Water is the most abundant compound in the body, and most of life’s essential processes take place in this aqueous environment. Although there is substantial variation, the total body water (TBW) of most domestic animals is approximately 60% of body weight (0.60 l/kg).Electrolytes are substances that exist as positive or negative charged particles in aqueous solution. The positively charged particles are “cations,” and the negatively charged particles are “anions”. The osmotic properties of a solute in solution are related to the number of particles in solution and not to its weight or its charge. One osmole of a non-dissociable substance is equal to its molecular weight in grams. One osmole of any substance that dissociates in solution into two or more particles is equal to the molecular weight in grams divided by the number of particles into which each molecule dissociates. Osmolarity is defined as the number of osmoles per l of final solution, whereas osmolality is the number of osmoles per kilogram of water. Although the expressions are similar, osmolality more correctly describes the osmotic properties as measured in the clinical laboratory.

Biotechnology is being harnessed in various aspects of the livestock industry to hasten breed development for improved animal health and welfare, enhanced reproduction, and improved nutritional quality and safety of animal-derived foods. Various biotechnology methods are used in improving the breeding stock of animals. These include artificial insemination (AI), embryo transfer (ET), in-vitro fertilization (IVF), sexing of semen and embryos, micromanipulation of oocytes/ embryos, transgenesis, somatic cell nuclear transfer and the emerging technology on genomics and marker-assisted selection (MAS). Artificial Insemination: One of the earliest perfected technology is artificial insemination (AI) where new breeds of animals are produced through the introduction of the male sperm from one superior male to the female reproductive tract without mating. AI reduces transmission of venereal disease, lessens the need of farms to maintain breeding males, facilitates more accurate recording of pedigrees, and minimizes the cost of introducing improved genetics. Various technologies have evolved that led to the efficient use of AI in developing desired livestock, including the methods of freezing semen or cryopreservation and sperm sexing.

Introduction

Cell culture is a technique that involves the isolation and in vitro maintenance of cells isolated from tissues or whole organs derived from animals, microbes or plants. This technique has immeasurable role in the field of biology. The scientific progress in biology, microbiology and biotechnology in recent years can be credited on the development of cell culture technology. With the development of cell culture the isolation of viruses has become less cumbersome, the assessment of the efficacy and toxicity of new drugs has been easier, new vaccines are being developed and there has been development in biotechnology assisted reproductive technology.

In general, animal cells have more complex nutritional requirements and usually need more stringent conditions for growth and maintenance. For the growth of the cell in vitro specific mediums are required by the cells. Previously natural mediums like plasma ,serums were used but with the advent of synthetic media the specific needs of different cell types can be catered with. Cell culture is governed by a provided set of protocols requiring a sterile pure culture of cells, various nutrients for its growth, sterile cell culture hood, appropriate aseptic techniques and the utilisation of suitable conditions for optimal viable growth of cells.

Introduction

Biochemical techniques are fundamental tools that allow scientists to investigate the molecular intricacies of biological systems, including DNA, RNA, and proteins. These techniques are essential for understanding cellular processes, genetic functions, and disease mechanisms. By employing biochemical methods, researchers can dissect complex biological systems at the molecular, cellular, and genomic levels, paving the way for advances in basic biology and therapeutic interventions.

Introduction

In a historic landmark, Genentech, a young Californian Company pioneering in gene splicing (which includes recDNA technology, genetic engineering, gene transplantation, gene cloning), saw its stock price skyrocket form $35 to a staggering $89 within just 20 minutes of the New York Stock Exchange’s opening bell on October 15, 1980. This was marked as the fastest stock price growth ever witnessed on that exchange. The company’s success was attributed to the ground-breaking work it had been involved in producing human insulin through genetic engineering that offered a revolutionary alternative for diabetics allergic to the then traditional insulin of porcine origin. Genentech’s pioneering work in gene splicing has its roots in the early 1970s. In 1972, Stanley Cohen and Herbert Boyer were credited with creating the first recombinant DNA molecule. Their experiments demonstrated the ability to create recombinant DNA molecules that could be introduced into bacteria like E? coli, offering a foundation for future advancements in genetic engineering.

Introduction

Nucleic acids, primarily DNA and RNA, are essential for the storage and expression of genetic information. Under physiological conditions, the amino and oxo tautomers of purines and pyrimidines predominate. Adenine a., guanine (G), cytosine c., thymine (T), and uracil (U) are the main building blocks of nucleic acids. Other building blocks include 5-methylcytosine, 5-hydroxymethylcytosine, pseudouridine, and N-methylated heterocycles. A -glycosidic bond connects D-ribose or 2-deoxy-D-ribose to the N-1 of a pyrimidine or the N-9 of a purine, with the syn conformers being the most common.

Nucleotides are the monomeric units of nucleic acids that contain a phosphate group attached to the sugar’s 3’ or 5’ hydroxyl. Extra phosphate groups form nucleoside diphosphates and triphosphates. These have high group transfer potential and help make covalent bonds. Cyclic phosphodiesters cAMP and cGMP act as second messengers. 3′ 5′ phosphodiester bonds link nucleotides to form polynucleotides, which have a directional 5′ and 3′ end. Four bases—A, G, C, and T—compose DNA, and hydrogen bonds (A to T, G to C) hold them together to form a double helix. Human DNA consists of approximately 3 billion base pairs organized into 23 chromosomes, encoding around 25,000 protein-coding genes and various non-coding RNAs (ncRNAs).

Introduction

Robert Brown discovered the nucleus in some plant cells in 1831. After analyzing tissues from a range of animals and plants, T. Schwann (1839) declared, “All living organisms are composed of cells.”

The fundamental elements of Prokaryotic and Eukaryotic cells

The simplest cells, called prokaryotic cells, have a straightforward structural arrangement. There is only one membrane system in it. They contain rickettsia, spirochete, mycoplasma, virus, blue-green algae, and bacteriablue-green algae, or cyanobacteria are the largest and most intricate prokaryotes, where higher plant photosynthesis occurs and have changed throughout time.

Prokaryotic cells

Prokaryotic cells range in size from 1 to 10 µm. They take place in a range of formats. A bacterial cell is made up of three basic parts:

I. External layer: It is composed of the inner cell or plasma membrane, middle cell wall and outer slimy capsule.

a. Cell membrane: The thin, flexible membrane of the cell, composed of proteins and lipids, regulates the flow of chemicals throughout the cell. It carries respiratory enzymes for reactions that release energy. Respiratory enzymes are found in mesosomes, which are folded regions of the plasma membrane that are compared to the mitochondria in eukaryotic cells. Similarly, the pigments and enzyme molecules found in photosynthetic cells are linked to the in-folds of the plasma membrane known as photosynthetic lamella, which is responsible for absorbing light and converting it into chemical energy.

b. The cell wall: It is the non-living, semi-rigid layer that encloses the cell membrane, with a thickness that varies from 1.5 to 100 µm. It is chemically made up of peptidoglycans. Certain bacteria, such as mycoplasma, don’t have cell walls.

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

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

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

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

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.

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.