GOMAL UNIVERSITY D.I.KHAN To Study the Effect of Salts and Complexes of Palladium and Vanadium Metals on the Status of Thiols in Blood Components Pharmacological and Toxicological Perspectives BY: Mohammad Mukhtiar Thesis Submitted To the Department of Pharmaceutical Chemistry Faculty of Pharmacy in Partial Fulfillment of Requirements for the Degree of DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACEUTICAL CHEMISTRY, FACULTY OF PHARMACY GOMAL UNIVERSITY DERA ISMAIL KHAN (KPK) PAKISTAN Jun 2013 DEDICATION To my Parents Who took Pains to Make the Way Easy for me towards this Success The work presented in this dissertation, has been carried out by me at the Laboratory of Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gomal University D.I. Khan and Department of pure and Applied Chemistry University of Strathclyde, Glasgow Scotland UK Under the supervision of PROF. DR. MUHAMMAD FARID KHAN Mohammad Mukhtiar [email protected] CERTIFICATE OF APPROVAL “To Study the Effect of Salts and Complexes of Palladium and Vanadium Metals on the Status of Thiols in Blood Components . Pharmacological and Toxicological Perspectives Thesis prepared by Mr. Mohammad Mukhtiar under my guidance in partial fulfillment of the requirements of the degree DOCTOR OF PHILOSOPHY IN PHARMACY is hereby approved for the submission to the Faculty of Pharmacy, Gomal University D.I.Khan. PROF. DR. MUHAMMAD FARID KHAN Supervisor Department of Pharmaceutical Chemistry Faculty of Pharmacy, Gomal University Dera Ismail Khan ROLL NO 20 SESSION 2008-2012 RESEARCH SUPERVISOR AND PROF. DR. MUHAMMAD FARID KHAN INTERNAL EXAMINER Department of Pharmaceutical Chemistry Faculty of Pharmacy CHAIRMAN Mr. Ghulam Mustafa Department of Pharmaceutical Chemistry Faculty of Pharmacy DEAN Mr. NUSRAT ULLAH Dean Faculty of Pharmacy EXTERNAL EXAMINER ____________________________ Ph.D Scholar Mohammad Mukhtiar (B. Pharm.) Table of Contents S.No Contents Page Acknowledgment 1 Abstract 2 Chapter:1 General introduction 1.1. Background 4 1.2. Thiols 4 1.3. Glutathione 6 1.3.1. History of Glutathione 6 1.3.2. Introduction 6 1.3.3. Protective functions of glutathione 9 1.3.5. Glutathione synthesis 14 1.3.6. Pharmacokinetics 17 1.3.7. Measurement of Glutathione 22 1.4. N‐ Acetyl cysteine (NAC) 22 1.4.1. Introduction 22 1.4.2 Biochemistry and pharmacokinetics 23 1.4.3. Clinical indications 23 1.5. D‐penicillamine (DPA) 24 1.5.1. Pharmacological profile 25 1.5.2. Biological role of D‐penicillamine 25 1.6. Albumin 26 1.6.1. Introduction 26 1.6.2. Medical uses 26 1.7.1. Vanadium 26 1.7.2. Chemistry and compounds 26 1.7.3. Natural occurrence of Vanadium 27 1.7.4. Biochemistry 28 1.7.5. Pharmacokinetics 28 1.7.6. Biological role of Vanadium 28 1.7.7. Pharmacological and therapeutic importance of Vanadium 29 1.7.8. Toxicological profile of Vanadium compounds 30 1.8.1. Palladium 31 1.8.2. Natural Occurrence and Environmental level 32 1.8.3. Palladium Compounds 32 1.8.4. Pharmacokinetics 33 1.8.5 Therapeutic Uses 34 1.8.6 Toxicology of Palladium 34 1.9.1. Scientific objectives 35 CHAPTER: 2 UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium compounds on The Status of Thiols (Glutathione, N‐ Acetyle cysteine and D‐penicillamine) in Aqueous Medium 2.1. Introduction 36 2.2. Methodology 37 2.2.1. Materials 37 2.2.2. Method 37 2.2.2.1. Preparation of stock solutions 37 2.2.2.2. Estimation of GSH by Ellman’s (Modified) Method 39 2.2.2.3. Standard Calibration Curve 39 2.2.2.4. Experimental Protocol 40 2.3. Results 41 2.3.1. Standard Curve of Glutathione N‐Acetyle Cysteine and D‐Penicillamine 41 2.3.2. Palladium Results 43 2.3.3. Effect of various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical 43 Status Glutathione and With Time (0 to90 Minutes) 2.3.4. Effect of Two Conc. (6.7µM and 67µM) of PDN on the Chemical Status 45 Glutathione with pH (7.0, 7.6, 8.0) 2.3.5. Effect of various Conc. (6.7µM to 67µM) of PDN/BBNPDC on the 45 Chemical Status N‐ Acetylcysteine and With Time (0 to90 Minutes) 2.3.6. Effect of Two Conc. (6.7 and 67µM) of PDN on the Chemical Status N‐ 47 Acetylcysteine with PH (7.0, 7.6, 8.0) 2.3.7. Effect of various Conc. (6.7 to 67uM) Conc. PDN/ BBNPDC on the 47 Chemical Status D‐Penicillamine and With Time (0 to 90 Minutes) 2.3.8. Effect of Two Conc. (6.7 and 67uM) of PDN on the Chemical Status of D‐ 49 Penicillamine with pH (7.0, 7.6 and 8.0) 2.4. Results of Vanadium Metal 49 2.4.1. Effect of various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical 49 Status Glutathione and with time (0 ‐90 Minutes) 2.4.2. Effect of Two Conc. (6.7 and 67uM) (AMV) on the Chemical Status 51 Glutathione, with pH (7.0, 7.6 and 8.0) 2.4.3. Effect of various Conc. (6.7µM to 67µM) of AMV /VOTEO on the Chemical 52 Status N‐ Acetylcysteine, and with Time (0 to 90 Minutes) 2.4.4. Effect Two Conc. of (6.7 to 67µM) of AMV on The Chemical Status N‐ 53 Acetyle Cysteine (NAC) with pH (7.0, 7.6, 8.0) 2.4.5. Effect of Various Conc. (6.7µM to 67µM) of AMV/ VOTEO on the Chemical 54 Status D‐Penicillamine, and with time (0 to 90 Minutes) 2.4.6. Effect of Two conc. (6.7 and 67uM) of (AMV) on the Chemical Status D‐ 55 Penicillamine, with pH (7.0, 7.6, 8.0) 2.5. Discussion 56 CHAPTER: 3 UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium on The Status of Thiols (Glutathione) in whole Blood (plasma and Cytosolic fraction) 3.1. Introduction 60 3.2. Methodology 61 3.2.1. Materials 61 3.2.2. Method 61 3.2.2.1. Preparation of Stock Solutions 61 3.2.2.2. Isolation of Blood components 63 3.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method 63 3.2.2.4. Experimental Protocol 64 3.3. Results 65 3.3.1. Results in Blood Plasma 65 3.3.1.1. Effect of various Conc. (6.7 to 67µM) of PDN/ BBNPDC on the Chemical 65 Status Plasma Glutathione, and with time (0 to90 Minutes) 3.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on The Chemical Status Plasma 68 Glutathione, with pH (7.0, 7.6, 8.0) 3.3.1.3. Effect of Various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical 68 Status of Plasma Glutathione, and With Time (0 to 90) Minutes 3.3.1.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status of 71 Plasma Glutathione with pH (7.0, 7.6, 8.0) 3.3.2. Results in Cytosolic Fraction (C.F) 72 3.3.2.1. Effect of Various Conc. (6.7 to 67µM) of (PDN / (BBNPDC) on the 72 Chemical Status of C.F Glutathione, and With Time (AT 0 ‐90 Minutes) 3.3.2.2. Effect of Two Conc. (6.7µM and 67µM) Of PDN on the Chemical Status of 75 C.F Glutathione with pH (7, 7.6, 8.0) 3.3.2.3. Effect of various Conc. (6.7 to 67µM) Of (AMV)/ (VOTEO) On the Chemical 76 Status C.F Glutathione, and With Time (At 0 ‐90 Minutes) 3.3.2.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status C.F. 79 Glutathione with pH (7.0, 7.6, 8.0) 3.4. DISCUSSION 80 CHAPTER: 4 UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium on the Status of Thiols (Glutathione) in WBC, S 4.1. Introduction 82 4.2. Methodology 83 4.2.1. Materials 83 4.2.2. Method 83 4.2.2.1. Preparation of stock solutions 83 4.2.2.2. Isolation of WBC Component (Lymphocytes (T‐cell and B‐cells) and 85 Neutrophils 4.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method 87 4.2.2.4. Reaction Protocol 87 4.3. Results 88 4.3.1. Results of Lymphocytes with Palladium and Vanadium Compounds 88 4.3.1.1. Effect of Various Conc (6.7 to 67µM) Of PDN / BBNPDC on the Chemical 88 Status of Lymphocytes Glutathione, and with Time (0 to ‐90 Minutes) 4.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on the Chemical Status 89 Lymphocytes Glutathione with PH (7, 7.6, 8.0) 4.3.1.3. Effect of Various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical 90 Status of B‐Cells Glutathione, and with time (0 ‐90) Minutes 4.3.1.4. Effect of Two Conc. (6.7 to 67uM) PDN on the Chemical Status of B‐Cells 91 Glutathione with pH (7, 7.6, 8.0) 4.3.1.5. Effect of Various Conc. (6.7 to 67uM) PDN on the Chemical Status of T‐ 92 Cells Glutathione, and with Time (at 0 ‐90 Minutes), 4.3.1.6. Effect of Two Conc. (6.7 and 67uM) PDN on the T‐Cells Chemical Status 94 Glutathione with pH (7, 7.6, 8.0) 4.3.1.7. Effect of various Conc. (6.7 to 67µM) AMV on The Chemical Status of 94 Lymphocytes Glutathione and with Time (0‐90) 4.3.1.8.