A PRACTICAL MANUAL IN BIOCHEMISTRY & CLINICAL BIOCHEMISTRY
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2 A PRACTICAL MANUAL IN BIOCHEMISTRY & CLINICAL BIOCHEMISTRY UNIVERSITY OF PAPUA NEW GUINEA SCHOOL OF MEDICINE AND HEALTH SCIENCES DIVISION OF BASIC MEDICAL SCIENCES DISCIPLINE OF BIOCHEMISTRY & MOLECULAR BIOLOGY 4 th EDITION of PRACTICAL MANUAL IN BIOCHEMISTRY Victor J. Temple, RACHAEL ROWE, Nigani Willie & Samson Grant Port Moresby University of Papua New Guinea Press 2013 Discipline of Biochemistry & Molecular Biology Page 1
3 This Practical Manual in Biochemistry & Clinical Biochemistry is a modified and updated version of the Practical Manual in Biochemistry by Dr. G.R.E. Swaniker, Dr. A. C. Banerjee and Mrs. S. Smillie. The 2 nd Edition of this manual was edited in 1992 by Dr. J. G. O. Harding and Mrs. B. R. Alfred. The 2 nd Edition was also reprinted in The 3 rd Edition was a modified and updated version of the 2 nd Edition by Assoc Prof. Victor J. Temple, Mr. Nigani Willie and Mr. Samson Grant. This 4 th Edition is an updated version of the 3 rd Edition. This 4 th Edition is an updated and greatly modified version of the 3 rd Edition. 4 th Edition: A Practical Manual in Biochemistry & Clinical Biochemistry ISBN: Edited by: Assoc. Prof. Victor J. Temple, Ms Rachael Rowe, Mr. Nigani Willie & Mr. Samson Grant Published by UPNG Press and Uni-Bookshop P.O. Box 413, University PO, UPNG Campus, Waigani, N.C.D, Port Moresby PNG upngbooks@gmail.com ACKNOWLEDGEMENTS The editors would like to acknowledge the undergraduate students in the School of Medicine and Health Sciences (SMHS) University of Papua New Guinea (UPNG) for their enthusiasm and commitment to the study of Biochemistry and Molecular Biology. We acknowledge Ms Jennie Bautau-Grant in the Biochemistry laboratory, for her dedicated service and hard work in keeping the laboratory fully functional at all times. Many thanks also to the technical officers in BMS. We thank all our colleagues in the Division of Basic Medical Sciences (BMS) for their support. We are extremely grateful to Ms Theresa Dunamb, the secretary in BMS, for providing the electronic copy of the 2 nd edition of this manual. Constructive comments and recommendations for improvement in the next edition of this manual are most welcome. Discipline of Biochemistry & Molecular Biology Page 2
4 # CONTENT Page # 1 Table of content Chapter 1: Introduction: Safety in the Biochemistry Laboratory: Guidelines and Precautions: Practical Reports & Assignments Chapter Structure of water; Water as a major solvent Section one: Solutions: Molarity (Molar concentration) Normality Percent-of-solute; Percent based on weight Osmolarity SECTION TWO: Dilutions; Simple and Serial dilutions: Practical Session One: Preparation and Dilution of Solutions Chapter SECTION ONE: The ph scale; Ionic Product of Water (Kw): Significance of the Ion Product of Water: Relationship between the Ionic Product (Kw) of water and the ph scale SECTION TWO: Acids, Bases and Buffers Strength and Concentration of acidic and basic solutions; Bronsted and Lowry concepts of acids & bases (Conjugate Acid Base Pair) Buffers, Range of Buffer and Buffer Capacity: Handerson-Hasselbalch equation; How does a buffer work? The ph range of a buffer solution; Buffer Capacity; The ph METER Practical Session Two: Measurement of ph; using ph Meter Practical Session Three: Section A: Buffer Solutions Practical Session Three: Section B: Saliva Chapter Colorimetry and Spectrophotometry Transmittance; Absorbance; Lambert s Law; Beer s Law; Beer-Lambert s equation Molar Extension Coefficient; Limitations of Beer-Lambert s Equation Colorimeter and Spectrophotometer Practical Session Four: Colorimetry and Spectrophotometry Chapter Estimation and Separation of Proteins in Blood Section One: Estimation of Serum / Plasma Proteins Section Two: Separation of Serum Proteins Practical Session Five: Estimation of Proteins in Blood Practical Session Six A: Separation of Proteins using cellulose strips Practical Session Six B: Separation of serum proteins in 1.0% Agarose Gel Chapter Six Chromatography: Absorption, Affinity, Gas-liquid, Gel-filtration or molecular sieving, Partition chromatography Practical Section Seven: Paper Chromatography of Amino Acids in Urine Practical Session Eight: Semi-quantitative assessment of Aspirin by Thin Layer Chromatography (TLC) Chapter Seven Estimation of Amylase Activity in Saliva and Serum Practical Session Nine: Estimation of Salivary Amylase Activity Chapter Eight Blood Glucose Practical Session Ten: Determination of Glucose in Serum Chapter Nine Estimation of Serum Total Cholesterol Practical Session Eleven: Estimation of Cholesterol in Serum Chapter Ten Discipline of Biochemistry & Molecular Biology Page 3
5 53 Renal (Kidney) Function I: Practical Session Twelve: Urinalysis Part I: Section I: Using Multistick Section II: Physical Examination of Urine Determination of Specific Gravity of Urine Section III: Chemical Analysis of Urine Tests for Proteins in Urine Tests for Glucose in Urine: Test for Ketone Bodies: Rothera s Test: Test for Bilirubin & Urobilinogen in Urine Practical Session Thirteen: Urinalysis Part II Microscopic Examination of Urine Chapter Eleven Renal (Kidney) Function II: Determination of Serum Urea: Practical Session Fourteen: Estimation of Urea in Blood (Serum) Chapter Twelve Renal (Kidney) Function III: Creatinine Clearance Practical Session Fifteen: Creatinine Clearance Chapter Thirteen Renal (Kidney) Function IV: Urinary Stones (Nephrolithoisis or Renal Calculi) Practical Session Sixteen: Estimation of Uric Acid in Urine and Serum: Section 1: Procedure for Determination of Uric Acid in Urine: Section II: Determination Of Uric Acid In Serum: Chapter Fourteen Liver Function Tests: Functions of the Liver: Biochemical Parameters in LFT Diagnostic significance of AST, ALT, ALP, GGTP Total Protein (Albumin and Globulins) Practical Session Seventeen: Estimation of Serum Bilirubin Types of jaundice: Haemolytic or Pre-hepatic; Hepatic; Post-hepatic Estimation of Bilirubin in Serum Practical Session Eighteen: Serum Alkaline Phosphatase Practical Session Nineteen: Estimation of serum ALT and AST Chapter Fifteen Diabetes Mellitus (DM): Practical Session Twenty: Oral Glucose Tolerance Test and Urinalysis Chapter Sixteen: Molecular Biology Practical Session Twenty One: Extraction of DNA Chapter Seventeen: Assignments References Appendix Discipline of Biochemistry & Molecular Biology Page 4
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