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I

PHARMACOLOGICAL EVALUATION OF

DIFFERENT COMBINATIONS OF POTASSIUM

NITRATE AND SULPHURIC ACID AGAINST

ETHYLENE GLYCOL-INDUCED UROLITHIASIS

A thesis submitted in partial fulfillment of the requirements for

the degree of Master of Philosophy (M.Phil.) in the subject of

PHARMACOLOGY

By

Muneeb ur Rehman

977/IU.M.Phil/2018

(SESSION 2018-2020)

Department of Pharmacology

Faculty of Pharmacy

The Islamia University of Bahawalpur

VI

“From the core of heart, my work is sincerely dedicated to

my Parents, Teachers, Family, Friends, and to Each &

Every person who has been in contact with me at any stage

of my life.”

VII

Table of Contents

Student’s Declaration .......................................................... Error! Bookmark not defined.

Supervisor’s Certificate .................................................................................................. III

Certificate ............................................................................. Error! Bookmark not defined.

Table of Contents ........................................................................................................... VII

List of Tables ..................................................................................................................... X

List of Figures .................................................................................................................. XI

List of Abbreviations ..................................................................................................... XII

Acknowledgments ........................................................................................................ XIII

Abstract ......................................................................................................................... XIV

1. Introduction ................................................................................................................... 1

2. Literature Review .......................................................................................................... 3

2.1.Urolithiasis .................................................................................................................... 3

2.1.1. Pathophysiology of Urolithiasis .............................................................................. 3

2.1.2. Saturation and Crystallization ............................................................................... 4

2.1.3. Crystals Formation .................................................................................................. 5

2.1.3.1.Nucleation ............................................................................................................... 5

2.1.3.2.Aggregation ............................................................................................................ 5

2.1.3.3.Crystal Growth....................................................................................................... 5

2.1.4. Sites of Stone Growth .............................................................................................. 5

2.1.4.1.Randall’s Plaque .................................................................................................... 5

2.1.4.2.Calcium Oxalate Receptors in Collecting Duct Epithelium ............................... 6

2.1.5. Promoters of Stone Formation .............................................................................. 6

2.1.5.1.Uric acid or Urate .................................................................................................. 6

2.1.5.2.Urine pH ................................................................................................................. 7

2.1.6. Inhibitors of Stone Formation ................................................................................ 7

2.1.6.1.Alkaline pH ............................................................................................................. 7

2.1.6.2.Citrate ..................................................................................................................... 7

2.1.6.3.Pyrophosphate ........................................................................................................ 8

2.1.6.4.Phytate .................................................................................................................... 8

2.1.6.5.Magnesium.............................................................................................................. 8

2.1.6.6.Glycoprotiens.......................................................................................................... 8

2.1.7. Types of Urinary Stones .......................................................................................... 8

2.1.7.1.Calcium Stones ....................................................................................................... 8

VIII

2.1.7.2.Uric Acid Stones ..................................................................................................... 9

2.1.7.3.Phosphate Stones .................................................................................................... 9

2.1.7.4.Cystine Stones......................................................................................................... 9

2.1.8. Risk Factors for urolithiasis ................................................................................... 9

2.1.8.1.Epidemiological Risk Factors ............................................................................. 10

2.1.8.2.Biochemical Factors ............................................................................................. 14

2.1.8.3.Genetics and Environmental Factors ................................................................. 16

2.1.9. Urolithiasis and Other Diseases ........................................................................... 16

2.1.10.Clinical Features of Urolithiasis .......................................................................... 16

2.1.11.Investigations of Urolithiasis ................................................................................ 16

2.1.12.Management of Urolithiasis ................................................................................. 17

2.1.12.1. Management of Acute Renal Colic ............................................................... 17

2.1.12.2. Treatment to Recurrent Stones .................................................................... 18

2.1.12.3. Surgical Treatment of Urolithiasis ............................................................... 19

2.2.Experimental Models of Urolithiasis ........................................................................ 19

2.3.Role of Minerals and Chemicals as Medicines ........................................................ 20

2.4.Potassium Nitrate ....................................................................................................... 21

2.5.Sulphuric Acid ............................................................................................................ 21

3. Research Methodology ................................................................................................ 23

3.1.Material and Equipment ........................................................................................... 23

3.2.Evaluation of Antiurolithiatic activity of Different Combinations of Potassium

nitrate and Sulphuric acid............................................................................................... 23

3.2.1. Animal Selection .................................................................................................... 23

3.2.2. Animal Model of Urolithiasis ............................................................................... 23

3.2.3. Urine Analysis ........................................................................................................ 24

3.2.3.1.Counting of Crystal.............................................................................................. 24

3.2.3.1.Urine Parameters ................................................................................................. 24

3.2.4. Serum Analysis ...................................................................................................... 25

3.2.5. Histology ................................................................................................................. 25

3.2.6. Statistical Analysis: ............................................................................................... 25

4. Results and Discussion ................................................................................................ 26

4.1.Ethylene Glycol-Induced Model of Urolithiasis ...................................................... 26

4.1.1. The Effects of Different Combinations of Potassium nitrate and Sulphuric

acid on Percent change in Bodyweight........................................................................... 27


acid on Various Urine Parameters ................................................................................. 27

4.1.2.1. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on

Crystal Count ................................................................................................................... 30

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Urine Volume ................................................................................................................... 30


Urinary pH ....................................................................................................................... 31


the Levels of Urinary Magnesium .................................................................................. 31


the Levels of Urinary Uric acid ....................................................................................... 32


acid on Various Serum Parameters............................................................................... 32


Serum Levels of Calcium................................................................................................. 32


Serum Levels of Phosphorous ......................................................................................... 43


Serum Levels of Creatinine ............................................................................................. 43


Serum Levels of Blood Urea Nitrogen (BUN) ............................................................... 43


acid on Histological Parameters ..................................................................................... 44

4.2.Conclusions of the Study ........................................................................................... 54

5. References .................................................................................................................... 55

X

List of Tables

Sr. No

TITLE

Page

No.

4.1.

The effects of different combinations of Potassium nitrate and

Sulphuric acid on % change in the bodyweight in EG/AC-induced

urolithiasis model in rats.

28

4.2.


Sulphuric acid on urinary crystal/mm3 in EG/AC-induced urolithiasis

model in rats.

33

4.3.


Sulphuric acid on urine volume (ml/24/h) in EG/AC-induced


35

4.4.


Sulphuric acid on urinary pH in EG/AC-induced urolithiasis model in

rats.

37

4.5.


Sulphuric acid on urinary magnesium levels in EG/AC-induced


39

4.6.


Sulphuric acid on urinary uric acid levels in EG/AC-induced


41

4.7.


Sulphuric acid on serum calcium levels in EG/AC-induced


45

4.8.


Sulphuric acid on serum phosphorous levels in EG/AC-induced


47

4.9.


Sulphuric acid on serum creatinine levels in EG/AC-induced


49

4.10.


Sulphuric acid on serum BUN levels in EG/AC-induced urolithiasis

model in rats.

51

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List of Figures

Sr. No

TITLE

Page

No.

4.1.


Sulphuric acid on % change in body weight in Ethylene glycol-

induced urolithiasis model in rats.

29

4.2.


Sulphuric acid on urinary crystals/mm3 in Ethylene glycol-induced


34

4.3.


Sulphuric acid on urinary volume (ml/24 h) in Ethylene glycol-


36

4.4.


Sulphuric acid on urinary pH in Ethylene glycol-induced urolithiasis

model in rats.

38

4.5.


Sulphuric acid on urinary levels of magnesium (mg/dl) in Ethylene

glycol-induced urolithiasis model in rats.

40

4.6.


Sulphuric acid on urinary levels of uric acid (mg/dl) in Ethylene


42

4.7.


Sulphuric acid on serum levels of calcium (mg/dl) in Ethylene glycol-


46

4.8.


Sulphuric acid on serum levels of phosphorous (mg/dl) in Ethylene


48

4.9.


Sulphuric acid on serum levels of creatinine (mg/dl) in Ethylene


50

4.10.


Sulphuric acid on serum levels of BUN (mg/dl) in Ethylene glycol-


52

4.11.


Sulphuric acid on Ethylene glycol-induced histological alterations in


53

XII

List of Abbreviations AC Ammonium Chloride

BC Before Christ

BUN Blood Urea Nitrogen

Ca Calcium

CaOx Calcium Oxalate

CC Creatinine Clearance

cm Centimeter

Conc. Concentration

CVD Cardiovascular Diseases

dl Deciliter

EG Ethylene Glycol

ESWL Extracorporeal Shock Wave Lithotripsy

G Gram

GFR Glomerular Filtration Rate

H Hour

H2SO4 Sulphuric acid

HCl Hydrochloric acid

i.p. Intra-peritoneal

K Potassium

Kg Kilogram

KSD Kidney Stone Diseases

L Liter

meq Milliequivalent

Mg Magnesium

mg Milligram

min Minutes

ml Milliliter

P Phosphorous

P.O Per Oral

PEAC Pharmacy Animal Ethics Committee

ROS Reactive Oxygen Species

rpm Revolutions Per Minute

SEM Standard Error of Mean

THP Tamm-Horsfall Protein

USA United State of America

UV Ultra Violet

v/v Volume by Volume

w/v Weight by Volume

WHO World Health Organization

$ Dollar

µl Microliter oC Degree Centigrade

XIII

Acknowledgments

In the name of ALLAH, the most Merciful and the most Beneficial. May Peace and

Blessings of ALLAH be upon His Prophet Hazrat Muhammad (peace be upon him).

All the praises be to ALLAH, The creator of the whole universe, who has conferred upon

me His countless blessings and endowed me to complete my M.Phil. Research work

successfully.

Special appreciation goes to my supervisor, Prof. Dr. Qaiser Jabeen (Professor and

Chairperson, department of Pharmacology, faculty of Pharmacy, the Islamia University of

Bahawalpur) for her supervision and constant support. Her invaluable help of constructive

comments and suggestions throughout the experimental and thesis work have contributed

to the success of this research.

I pay my gratitude to Prof. Dr. Naveed Akhtar (Dean, department of Pharmacy, Faculty

of Pharmacy, IUB) for his uncountable co-operation and support in the course of my

research work.

I would like to say special thanks to Mr. Tayyab (Store in-charge) who was extremely

kind and highly concerned to arrange all the required chemicals and other research related

material in quick time. Without his co-operation, it would have not been possible for me

to complete my research in time. Super thanks to Mr. Javed Iqbal and Mr. Muhammad

Ramzan (LA, Pharmacology research lab, IUB) for their support, guidance, co-operation

and the time they have spent with us during working as well as off-hours.

In the end, special thanks to all my M.Phil. Colleagues who have been helping me

directly or indirectly throughout the research work. Distinct thanks to seniors,

companions and juniors for giving me unforgettable memories. I would also pay deep

appreciations to my family for their love, motivation and financial support throughout the

study.

Muneeb ur Rehman

XIV

Abstract

Urolithiasis, the formation of stones in the kidney or urinary system, has affected

mankind from many decades and considered as one of the major problems throughout the

world. The main reason behind the urolithiasis is uneven distribution of minerals around

the world. With the advancement in the field of medicines, still the researchers are unable

to introduce a new molecular entity which can cure urolithiasis completely. However,

there are many preparations that are being used effectively for the treatment of

urolithiasis by local practitioners. The study was designed to validate the use of

preparations of Potassium nitrate and Sulphuric acid in various combinations.

Urolithiasis was induced by using ethylene glycol (EG) and ammonium chloride (AC) in

drinking water for twenty-one days. Control group was given distilled water throughout

the study while remaining groups were intoxicated with EG and AC for twenty-one days.

Then, all the animals were placed in metabolic cages and urine parameters were analyzed

for the confirmation of urolithiasis. Afterwards, positive control group was also treated

with distilled water for the next fourteen days. Treatment groups were treated with

different combinations of potassium nitrate and sulphuric acid; group I was administered

potassium nitrate (300 mg/100 ml) + Sulphuric acid (01 ml/100 ml), group II with

Potassium nitrate (300 mg/100 ml) + Sulphur (100 mg/100 ml), while, groups III and IV

were given dextrose (10%) in addition to the treatment as groups I and II, respectively.

After the end of the study (total 35 days), animals were placed in the metabolic cages for

twenty-four hours to collect the urine and each sample was analyzed for urinary crystals,

urinary volume, urinary pH and the levels of magnesium as well as uric acid. The animals

were anesthetized and blood was drawn by retro-orbital cavity to analyze the serum

parameters. Sera was obtained by centrifuging the blood at 4000 rpm for 10 minutes and

analyzed for the levels of blood urea nitrogen, calcium, creatinine and phosphorous by

using commercially available kits. Animals of each group were dissected out and one

kidney from the representative animal of each group was removed for histological

studies.

The results of treatment group II showed significant decrease in number of crystals

96.88±15.29 in comparison with day twenty-one value 142.50±3.39. At day thirty-five,

groups III and IV showed highly significant decrease 83.75±9.68 and 73.75±3.31/mm3 as

compared to their respective intoxicated values 130.36±3.96 and 160.25±3.23/mm3.

Decreased levels of calcium; i.e. 28.37±0.74, 25.36±1.12, 22.79±1.02 and 20.11±0.72

XV

mg/dl were found after the treatment with various potassium nitrate and sulphuric acid

combinations; i.e. I, II, III and IV in comparison with intoxicated group 29.61±0.99

mg/dl. The various combinations of potassium nitrate and sulphuric acid were found to be

effective in preventing as well as reversing the toxic changes produced by lithogenic

treatment such as loss in body weight, acidic pH, large volume of urine, lowered levels of

magnesium, increased urinary levels of uric acid, crystal count, raised serum blood urea

nitrogen, calcium, creatinine and phosphorous levels. The antiurolithiatic potential may

be due to the diuretic and urine alkalinizing property. The results rationalize the use of

these combinations against urolithiasis and further studies are recommended to explore

their exact mechanism(s) of action to serve as potential candidates against urolithiasis.

1

1. Introduction

Formation of stones in any organ of the body or duct is referred as lithiasis. Urolithiasis is

the process of stones formation in urinary tract and is one of the major problems for

mankind from many decades. Geographical data declares that this disease belongs to

Egyptian era and is the third most common disorder of the urinary system associated with

multiple risk factors which directly affects the patients quality of life (López and Hoppe,

2010, Thomas and Hall, 2005). The primary reason of urolithiasis is the unequal

distribution of the minerals around the world. Urinary stones are formed by the complex

formation of the minerals; i.e. calcium, oxalate and uric acid.

Worldwide prevalence of urolithiasis is an estimated lifetime risk of 8-15% in America

and Europe, 2-5% in Asia and around 20% in Middle East. In Pakistan, prevalence is

about 10-15%, primarily affecting fifty percent people of middle ages and sixty percent

children. Recurrence rate is also increasing which is estimated to be 10-23% in one year,

50% in next five year and more than 75% in next 20 years (Moe, 2006, Pak, 1998).

According to the ‘Urological Disease” survey in the United State of America, the annual

cost of the treatment of patient with urolithiasis in 1974 was estimated to be $47.2 million

which increased to $5.3 billion in 2000 (Moe et al., 2011). Economic impact explains the

importance for the development of cost effective treatment for the urolithiasis especially

in countries which are economically not so strong.

There are many extrinsic and intrinsic risk factors like age, gender, climate, water intake

and diet etc. Urolithiasis affects the people of all ages but people between ages 20 to 40

years are more prone. Urolithiasis affects men more as compared to women (3:1) which

may be due to nutritional as well as hormonal differences. Warm climates are related with

greater frequency of urolithiasis which may be due to increased sweating. High intake of

sodium and consumption of protein rich diet can increase the risk of urolithiasis. Higher

intake of water diluted the urine and decreases the chances of supersaturation with stone

forming constituents (Sellaturay and Fry, 2008, Abbagani et al., 2010, López and Hoppe,

2010). Urolithiasis is characterized by pain in loin that moves to groin and then to

scrotum. Blockage of urinary tract with stones causes slow, difficult and painful

urination. Urinary stones increase stay of microorganism in urinary system which can

infect the already damaged epithelium (Parmar, 2004, Thomas and Hall, 2005).

Many stone promoters, inhibitors and modifier have significant impact on the

development, progression and prevention of disease. The stone inhibitors; i.e.

2

magnesium, citrate, glycosaminoglycans and osteopontin inhibit the stone formation

naturally (Ratkalkar and Kleinman, 2011). Supersaturation of urine with stone forming

elements is the main step which leads towards nucleation, aggregation and growth of

crystals. Experimental models have shown that CaOx crystals causes epithelial injury of

renal tubules which is accompanied by lipid peroxidation as well as free radical

production by the generation of Reactive Oxygen Species (ROS) (Brown and Evans,

1998, Worcester and Coe, 2008, Abbagani et al., 2010).

Diagnostic investigation may involve history of patient, physical examination, blood,

urine and stone analysis and different types of imaging techniques; e.g. ultrasonography,

radiography, isotope renography, contrast and non-contrast computerized tomographic

scan(CT scan) (Thomas and Hall, 2005). Management of urolithiasis includes

conventional measures, pharmacological and non-pharmacological treatments.

Conventional measures include increased intake of citrus fruits and decreased intake of

animal proteins. Pharmacological treatment modify certain factors which have impact on

the formation of urinary stones; e.g. Hypercalciuria can be treated with thiazide diuretics,

potassium citrate is used to increases the urinary excretion of citrate and allopurinol is

effective in the treatment of hyperuricosuria,. Few decades ago surgical removal of the

stone (lithotomy) was the only management, but due to greater risk of complications like

infections related with surgery and hemorrhage, it was no more considered (Thomas and

Hall, 2005, López and Hoppe, 2010). Extracorporeal shock wave lithotripsy, uretero-

renoscopy, percutaneous nephrolithotomy and endoscopic lithotomy are the latest

techniques of current era that are used to manage the urolithiasis (Ratkalkar and

Kleinman, 2011). These invasive techniques have increased risk of infections, so there is

a need of further investigations which may lead to new pharmacological entity in the

treatment of urolithiasis.

In Pakistan, there are many mineral preparations that are being practiced effectively in the

treatment of urolithiasis by local practitioner. Folk history reveals that chemical and

minerals play a vital role in the life and were used in the treatment of various diseases.

Minerals have been used for the curative purposes since early history. The purpose of the

study was to analyze the antiurolithiatic potential of combinations of potassium nitrate

and sulphuric acid against ethylene glycol-induced urolithiasis.

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