(FOR PARAMEDICAL STUDENTS) Poonam Bachheti Managing Director DPMI. Aruna Singh Principal DPMI

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2 BIOCHEMISTRY (FOR PARAMEDICAL STUDENTS) Poonam Bachheti Managing Director DPMI Aruna Singh Principal DPMI (An ISO 9001:2008 Certified Company) Vayu Education of India 2/25, Ansari Road, Darya Ganj, New Delhi

3 Biochemistry Copyright DPMI ISBN: First Edition: 2014 Price: 250/- All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the Author and Publisher. Printed & bound in India Published by: AN ISO 9001:2008 CERTIFIED COMPANY VAYU EDUCATION OF INDIA 2/25, Ansari Road, Darya Ganj, New Delhi Ph.: , Fax: vei@veiindia.com, vayueducation1@gmail.com Website:

4 PREFACE This textbook, Biochemistry is aimed at chemistry and biochemistry undergraduate students and first year biochemistry graduate students. It incorporates the lectures given to students with a strong chemistry background. An emphasis is placed on metabolism and reaction mechanisms and how they are studied. As the title of the book implies, the text lays the basis for an understanding of the fundamentals of biochemistry. Undergraduate biochemistry courses cover what proteins do, as enzymes, receptors, hormones, motors or structural components. The much more interesting question is to how proteins achieve all these functions? Presented here is an overview of the methods used in such projects, their possible applications, and their limitations. The technical help rendered by Dr. Pallavi Srivastava in giving final shape to the, manuscript is duly acknowledged. We have done our best to make it a complete book and hope the students will get a good exposure to clinical biochemistry basic theory and practical. We also want to thank the many students and professional colleagues who have offered their advice and constructive criticism throughout the development of this Book of Biochemistry (Authors)

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6 SYLLABUS BIOCHEMISTRY 1. INTRODUCTION TO BIOCHEMSTRY Definition, Introduction, Various types of laboratories, standardized clinical laboratory set up. 2. ETHICS IN LABORATORY Principles of ethics, Responsibilities of Laboratory workers, Role of medical Lab. Technologist. 3. SAFETY MEASURES Safety rules, fire extinguisher, storage of chemicals, Universal safety Precautions 4. WASTE DISPOSAL House hold waste, Chemical waste, specimen waste, sharp waste. 5. FIRST AID First aid measures First aid kit

7 viii Biochemistry 6. TOTAL QUALITY MANAGEMENT Introduction(Quality control, Quality leadership, Quality improvement) Quality assurance, Accuracy, Precisie, Sensitive Specific. 7. BASIC STATISTICS Use of standard deviation in the laboratory. Preparation of Quality control chart. Levy Jenning chart Linearty 8. GLASSWARES General glassware Care and maintenance of glassware 9. UNIT OF MEASUREMENT SI unit (International systems of unit) Measurement of volume Molarity, Molality, Normality, Mole fraction, Mass percentage. 10.CALIBRATION Basic for calibration Procedure for calibration of volumetric flask Procedure for calibration of volumetric pipette Nominal and true values, To contain, To deliver.

8 Contents ix 11.SOLUTIONS AND REAGENTS Introduction, Preparation of Reagents, Solutions, Standard solution Primary standard, Secondary standard. Stock solution, Working solution, Osmosis, Diffusion 12.PREPARATION OF NORMAL HYDROCHLORIC ACID Procedure, Calculation 13.PREPARATION OF NORMAL SALINE SOLUTION Procedure, Calculation 14.PREPARATION OF VARIOUS DILUTIONS FROM A STOCK SOLUTION Procedure, Requirements 15.PREPARATION AND STANDARDIZATION OF SOLUTION Introduction Atoms, Elements, molar solution, Molal solution, Normal solution Avogadro number, Percentage of solution W/V & V/V. 16.PREPARATION OF PROTEIN FREE FILTRATE Principle, Importance, Procedure use. 17.BUFFER SOLUTIONS Types of Buffers Buffering against PH changes in Biological systems. 18.pH ANALYSIS Introduction of ph, PH meter, Principles of operating a ph meter, weak acids and bases, analysis of ph, calibration of PH meter.

9 x Biochemistry 19.ACIDS & BASES Introduction Chemistry of acids and bases titration 20.INDICATORS Introduction Types of indicators universal Indicator Uses of Indicators 21.SPECIMEN COLLECTION Basic steps for drawing a blood samples Deciding specimen types, Anticoagulants, Vacutainers, Specimen Stability-Collection of urine samples. 22.INSTRUMENTS I Introduction,Balances, Hot Plates and Magnetic stirrer, Hot air oven, Incubators, Centrifuge, Water bath( Principle, Use, Operating Instruction) 23.INSTRUMENTS II Colorimeter, Photometer Laboratory tests based on colorimeter and photometer Standardization of photometer Selection of filter for determining intensity 24.TECHNIQUES IN BIOCHEMISTRY Electrophoresis, chromatography, Densitometer, Osmometry

10 Contents xi Distillation apparatus, Autoanalyzer based on immunoassay techniques Latest trends in automation Radial Immuno diffusion, Fluorescent antibody technique 25.BIOMOLECULES Introduction Chemical composition of biomolecules Metabolism, Catabolism, Anabolism, Metabolic diversity in living organism Law of thermodynamics 26.ROUTINE TESTS PERFORMED IN BIOCHEMISTRY Glucose(Sugar fasting,glucose PP, Glucose Random) Liver function test(bilirubin, Alkaline Phosphatase, SGOT, SGPT, Total protein,a/g Ratio,Albumin, Globulin) Lipid Profile test(cholesterol, Triglycerides, HDL,LDL,VLDL) Kindey function test(urea, Creatinine, uric acid,electrolyles) Cardiac Profile test(cpk,ldh,shbd) 27.NORMAL RANGE OF ROUTINE TESTS 28.CHEMISTRY OF CARBOHYDRATES

11 xii Biochemistry Carbohydrate introduction, Metabolism, Functions,Classification, Properties, Diabetes profile test. 29.DIABETIC PROFILE TESTS Introduction Determination of Blood glucose (sugar fasting, Post Prandial) (Principle, Procedure) Glycosylated Haemoglobin determination(principle, Clinical Significance, Procedure) Determination of urine glucose.

12 Contents xiii CONTENTS 1. Introduction to Biochemistry Ethics In Laboratory Safety & Precaution In Laboratory Waste Disposal First Aid Total Quality Management Basic Statistics Description of Glass Ware Unit of Measurement Calibration Solutions & Reagents Preparation of 1.0 N Hydrochloric Acid Solution Preparation of normal saline Working with Stock Solutions Standardization of A Sodium Hydroxide Solution Preparation of Protein Free Filtrate Buffer Solutions ph Analysis Acids & Bases Indicators Specimen Collection Instruments Colorimetery & Spectrophotometry Electrophoresis Biomolecules Routine Test performed in Biochemistry Diabetic profile Test Color Figures Index

13 CHAPTER 1 Introduction to Biochemistry BIOCHEMISTRY Biochemistry, sometimes called biological chemistry, is the study of chemical processes within, and relating to, living organisms. By controlling information flow through biochemical signaling and the flow of chemical energy through metabolism, biochemical processes give rise to the complexity of life. Over the last 40 years biochemistry has become so successful at explaining living processes that now almost all areas of the life sciences from botany to medicine are engaged in biochemical research. Today the main focus of pure biochemistry is in understanding how biological molecules give rise to the processes that occur within living cells, which in turn relates greatly to the study and understanding of whole organisms. Biochemistry is closely related to molecular biology, the study of the molecular mechanisms by which genetic information encoded in DNA is able to result in the processes of life. Depending on the exact definition of the terms used, molecular biology can be thought of as a branch of biochemistry, or biochemistry as a tool with which to investigate and study molecular biology. Much of biochemistry deals with the structures, functions and interactions of biological macromolecules, such as proteins, nucleic acids, carbohydrates and lipids, which provide the structure of cells and perform many of the functions associated with life. The chemistry of the cell also depends on the reactions of smaller molecules and ions. These can be inorganic, for example water and metal ions, or organic, for example the amino acids which are used to synthesise proteins. The mechanism by which cells harness energy from their environment via chemical reactions is known as metabolism.

14 2 Biochemistry LABORATORY ORGANIZATION Functional components of clinical laboratories The clinical laboratory can be attached to a hospital or it can be an independent laboratory. In either case, a pathologist is in charge of the laboratory. Who is awarded with the degree of medicine and specialized in Pathology. The medical laboratory technicians, on the other hand, holds a A degree or Diploma in MLT and receives 1 or 2 years of clinical laboratory training at lower level. The diagnostic results, obtained by the technologists or technicians, are entered into the request-report form of the phycians and are signed by the pathologist at the end of the day for external communication. All internal communications, however, will bear the signature of the technician preferably technologist with the date. ADMINISTRATOR PATHOLOGIST LABORATORY SPECIMEN COLLECTING AND PROCESSING HAMATOLOGY AND COAGULATION BIOCHEMISTRY BLOOD BANK MICROBIOLOGY Bacteriology Parasitology Mycology SEROLOGY CLINICAL PATHLOGY HISTOLOGY Cytology Fig. 1.1: Organization Structure of Clinical From the organizational point of view (fig 1.1) the hospital classifies the medical laboratory as a part of special diagnostic services. In order to function properly the laboratory is divided into several components. These components are artificial divisions made according to the convenience of the laboratory but they can be grouped differently, depending on the work load and number of employees. Ideally, the supervisor, who is a technologist will be in charge of the section and the technicians (bench-workers) will work under the supervision, of him communicate with external agencies, accomplish paper work, ordering of supplies, purchase of equipment, equipment maintenance, keeping of records, checking the inventory, providing current information to technicians and corresponding with pathologists and physicians. This ideal situation rarely exists in developing countries. Most technicians are on-the-job-trained (OJT) and in small laboratories they work single handed with probably one additional helper to perform non-technical work such as sterilization, distillation, preparation of swabs etc. Irrespective of organizational structures, the functional components of the clinical laboratory can be listed as follows.

15 Introduction to Biochemistry 3 1. Hematology 2. Blood bank (or immunohaematology) 3. Microbiology: a. Bacteriology b. Mycology c. Parasitology 4. Serology (or immunology) 5. Clinical pathology 6. Clinical biochemistry 7. Histology and cytology In some hospitals there is a team of phlebotomists, whose main function is to draw blood specimen from indoor and outdoor patients. Two laboratory floor plans are provided in the illustrations. The patients submit the request slip from the physician to register at the receiving desk. Following the registration appropriate specimen is collected and dispatched to the specific section for the laboratory testing. The laboratory may also have a receiving window which receives the specimen from the ward or outdoor blood drawing area. Hematology The hematology section of the laboratory is primarily involved in the microscopic examination of blood along with physical examination of whole blood and determination of hemoglobin. The diagnosis of blood-related disorder like anemia and leukemia largely depends on the hematological and peripheral blood film findings. The hematology laboratory also undertakes investigations for bleeding disorders. This involves a search for the missing coagulation factor that affects the clotting process. Other tests include determination of bleeding time, clotting time and platelet count. Blood Banking Blood Banking is also known as immnohaematology as it involves antigen antibody (immunologic) reaction which is associated with red blood cells. The primary function of the blood bank is to provide appropriate blood to the patient for transfusion. Transfusion of mismatched blood can be fatal. Because of the critical nature of this field, the technicians are expected to be highly proficient in performing blood compatibility testing. Transfusion reactions due to technical error do not commonly occur but clerical mistakes bring the higher risk. Hence, proper records and labelling are essential.

16 BioChemistry By Poonam Bachheti and Aruna Singh Publisher : Vayu Education ISBN : Author : Poonam Bachheti and Aruna Singh Type the URL : Get this ebook