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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
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“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.”
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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
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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
4.1.2. The Effects of Different Combinations of Potassium nitrate and Sulphuric
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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4.1.2.2. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Urine Volume ................................................................................................................... 30
4.1.2.3. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Urinary pH ....................................................................................................................... 31
4.1.2.4. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
the Levels of Urinary Magnesium .................................................................................. 31
4.1.2.5. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
the Levels of Urinary Uric acid ....................................................................................... 32
4.1.3. The Effects of Different Combinations of Potassium nitrate and Sulphuric
acid on Various Serum Parameters............................................................................... 32
4.1.3.1. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Serum Levels of Calcium................................................................................................. 32
4.1.3.2. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Serum Levels of Phosphorous ......................................................................................... 43
4.1.3.3. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Serum Levels of Creatinine ............................................................................................. 43
4.1.3.4. Effects of Different Combinations of Potassium nitrate and Sulphuric acid on
Serum Levels of Blood Urea Nitrogen (BUN) ............................................................... 43
4.1.4. The Effects of Different Combinations of Potassium nitrate and Sulphuric
acid on Histological Parameters ..................................................................................... 44
4.2.Conclusions of the Study ........................................................................................... 54
5. References .................................................................................................................... 55
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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.
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4.2.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary crystal/mm3 in EG/AC-induced urolithiasis
model in rats.
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4.3.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urine volume (ml/24/h) in EG/AC-induced
urolithiasis model in rats.
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4.4.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary pH in EG/AC-induced urolithiasis model in
rats.
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4.5.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary magnesium levels in EG/AC-induced
urolithiasis model in rats.
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4.6.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary uric acid levels in EG/AC-induced
urolithiasis model in rats.
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4.7.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum calcium levels in EG/AC-induced
urolithiasis model in rats.
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4.8.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum phosphorous levels in EG/AC-induced
urolithiasis model in rats.
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4.9.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum creatinine levels in EG/AC-induced
urolithiasis model in rats.
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4.10.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum BUN levels in EG/AC-induced urolithiasis
model in rats.
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List of Figures
Sr. No
TITLE
Page
No.
4.1.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on % change in body weight in Ethylene glycol-
induced urolithiasis model in rats.
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4.2.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary crystals/mm3 in Ethylene glycol-induced
urolithiasis model in rats.
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4.3.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary volume (ml/24 h) in Ethylene glycol-
induced urolithiasis model in rats.
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4.4.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary pH in Ethylene glycol-induced urolithiasis
model in rats.
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4.5.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary levels of magnesium (mg/dl) in Ethylene
glycol-induced urolithiasis model in rats.
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4.6.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on urinary levels of uric acid (mg/dl) in Ethylene
glycol-induced urolithiasis model in rats.
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4.7.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum levels of calcium (mg/dl) in Ethylene glycol-
induced urolithiasis model in rats.
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4.8.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum levels of phosphorous (mg/dl) in Ethylene
glycol-induced urolithiasis model in rats.
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4.9.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum levels of creatinine (mg/dl) in Ethylene
glycol-induced urolithiasis model in rats.
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4.10.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on serum levels of BUN (mg/dl) in Ethylene glycol-
induced urolithiasis model in rats.
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4.11.
The effects of different combinations of Potassium nitrate and
Sulphuric acid on Ethylene glycol-induced histological alterations in
urolithiasis model in rats.
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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
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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
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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
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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.
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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.
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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.