STUDIES ON CORRELATIONS BETWEEN URINE PARAMETERS AND FLUX VARIATIONS ON HUMAN URINE USING He-Ne LASER AND
ENCIRCLED FLUX ANALYSIS SYSTEM
by
AZRUL NIZAM BIN ALIAS
Thesis submitted in fulfilment of the requirements for the degree of Master of Science
January 2007
ii
ACKNOWLEDGEMENTS
I am very grateful to many people for the contributions they have made to this
research. I would also like to thank my supervisor, Prof. Madya Dr. Mohamad
Suhaimi Jaafar and co-supervisor, Dr. Khalid M. Omar Al-Hadithi for their
committed guidance and helpful discussions. I also owe a debt of gratitude to
Mr. Yahya, medical physics’s lab assistant for the excellent technical
assistance. I am also truly appreciate Universiti Sains Malaysia for giving me
financial assistance from Graduate Assistant Scheme and Penang Hospital
for the receipt of the patient urines. I would also like to thank my parents, Alias
bin Hussein and Hasnah bt. Mustapa for their never ending supports for me
and to my colleagues for their unstinting help throughout this research.
iii
TABLE OF CONTENTS
Page
Acknowledgements ii
Table of Contents iii
List of Tables vii
List of Figures x
List of Symbols xv
Abstrak xvi
Abstract xix
Chapter 1 : Introduction
1.1 Urine 3
1.2 Helium-Neon Laser 16
1.2.1 Flux of Laser 17
1.3 Objectives of the Research 18
1.4 Outline of Thesis 19
1.5 Literature Review 19
iv
Chapter 2 : Materials And Methods
2.1 Urine Samples 21
2.2 Instrumentation 21
2.3 Experimental Methods 22
Chapter 3 : Flux Analysis And Statistical Tests
3.1. Urine pH and Age 25
3.2 Urine pH and Urine Specific Gravity 26
3.3 Urine pH and Urine Protein 28
3.4 Urine pH and Urine Glucose 29
3.5 Urine Specific Gravity and Age 31
3.6 Urine Specific Gravity and Normal Urine 32
3.7 Urine Specific Gravity and Urine Glucose 33
3.8. Urine Specific Gravity and Urine Protein 35
3.9 Urine Protein and Age 36
3.10 Urine Glucose and Age 37
3.11 Flux and Age 38
3.12 Flux and Urine pH 45
3.13 Flux and Urine Specific Gravity 50
3.14 Flux and Normal Urine 55
v
3.15 Flux and Urine Glucose 80
3.16. Flux and Urine Protein 103
Chapter 4 : Summary and Conclusions 127
References 131
Appendices
Publication List
vi
LIST OF TABLES
Page
Table 1.1 The pH of urine compared with body fluids 10
and other material
Table 3.1 Normality Test (Kolmogorov-Smirnov) of flux peak 40
Table 3.2 Paired t-test of flux peak 40
Table 3.3 Normality Test (Kolmogorov-Smirnov) of total flux 42
Table 3.4 Paired t-test of total flux 42
Table 3.5 Normality Test (Kolmogorov-Smirnov) of flux peak 46
Table 3.6 Paired t-test of flux peak 46
Table 3.7 Normality Test (Kolmogorov-Smirnov) of total flux 48
Table 3.8 Paired t-test of total flux 48
Table 3.9 Normality Test (Kolmogorov-Smirnov) of flux peak 51
Table 3.10 T-Test of flux peak 51
Table 3.11 Normality Test (Kolmogorov-Smirnov) of total flux 53
Table 3.12 Paired t-test of total flux 53
Table 3.13 Normality Test (Kolmogorov-Smirnov) of flux peak 57
Table 3.14 Paired t-test of flux peak 57
Table 3.15 Normality Test (Kolmogorov-Smirnov) of total flux 58
Table 3.16 Paired t-test of total flux 58
Table 3.17 Normality Test (Kolmogorov-Smirnov) of flux peak 82
Table 3.18 Paired t-test of flux peak 82
Table 3.19 Normality Test (Kolmogorov-Smirnov) of total flux 83
Table 3.20 Paired t-test of total flux 83
vii
Table 3.21 Normality Test (Kolmogorov-Smirnov) of flux peak 105
Table 3.22 Paired t-test of flux peak 105
Table 3.23 Normality Test (Kolmogorov-Smirnov) of total flux 106
Table 3.24 Paired t-test of total flux 106
viii
LIST OF FIGURES
Page
Figure 1.1 Schematic diagram of a single nephron 5
Figure 1.2 Schematic of helium-neon laser. 17
Figure 2.1 Urine samples 23
Figure 2.2 He-Ne laser (0.95 mW) and Encircled Flux 23
Analysis System (EFAS) Model 8350, Photon Inc.
Figure 2.3 Schematic diagram of the experimental set-up. 24
Figure 3.1 Urine pH vs Age Range (years old) 26
Figure 3.2 Urine Specific Gravity (SG) vs Urine pH 27
Figure 3.3 Urine Protein vs Urine pH 29
Figure 3.4 Urine Glucose vs Urine pH 30
Figure 3.5 Urine Specific Gravity vs Age Range (years old) 32
Figure 3.6 Specific Gravity of Normal Urine 33
Figure 3.7 Urine Glucose vs Urine Specific Gravity (SG) 34
Figure 3.8 Urine Protein vs Urine Specific Gravity (SG) 35
Figure 3.9 Urine Protein vs Age Range (years old) 36
Figure 3.10 Urine Glucose vs Age Range (years old) 37
Figure 3.11 Flux peak obtained for males urine in different 38
age groups
Figure 3.12 Flux peak obtained for females urine in different 39 age groups
Figure 3.13 Total flux obtained for males urine in different 41
age groups
ix
Figure 3.14 Total flux obtained for females urine in different 41
age groups
Figure 3.15 Flux peak’s males and females urine vs age range 43
Figure 3.16 Total flux’s males and females urine vs age range 44
Figure 3.17 Flux peak of males and females urine vs urine pH 45
Figure 3.18 Total flux of males and females urine vs urine pH 47
Figure 3.19 Flux peak of males and females urine vs 50
urine specific gravity
Figure 3.20 Total flux of males and females urine vs 52
urine specific gravity
Figure 3.21` Point plot graph of flux peak (normal urine) of 55
males and females urine
Figure 3.22 Point plot graph of total flux (normal urine) of 56
males and females urine
Figure 3.23 2D Contour of Male (Normal Urine) 59
20-29 years old
Figure 3.24 3D Profile of Male (Normal Urine) 59
20-29 years old
Figure 3.25 Pattern for Male (Normal Urine) 60
20-29 years old
Figure 3.26 2D Contour of Male (Normal Urine) 60
30-39 years old
Figure 3.27 3D Profile of Male (Normal Urine) 61
30-39 years old
Figure 3.28 Pattern for Male (Normal Urine) 61
x
30-39 years old
Figure 3.29 2D Contour of Male (Normal Urine) 62
40-49 years old
Figure 3.30 3D Profile of Male (Normal Urine) 62
40-49 years old
Figure 3.31 Pattern for Male (Normal Urine) 63
40-49 years old
Figure 3.32 2D Contour of Male (Normal Urine) 63
50-59 years old
Figure 3.33 3D Profile of Male (Normal Urine) 64
50-59 years old
Figure 3.34 Pattern for Male (Normal Urine) 64
50-59 years old
Figure 3.35 2D Contour of Male (Normal Urine) 65
60-69 years old
Figure 3.36 3D Profile of Male (Normal Urine) 65
60-69 years old
Figure 3.37 Pattern for Male (Normal Urine) 66
60-69 years old
Figure 3.38 2D Contour of Male (Normal Urine) 67
70-79 years old
Figure 3.39 3D Profile of Male (Normal Urine) 67
70-79 years old
Figure 3.40 Pattern for Male (Normal Urine) 68
70-79 years old
xi
Figure 3.41 2D Contour of Female (Normal Urine) 68
20-29 years old
Figure 3.42 3D Profile of Female (Normal Urine) 69
20-29 years old
Figure 3.43 Pattern for Female (Normal Urine) 69
20-29 years old
Figure 3.44 2D Contour of Female (Normal Urine) 70
30-39 years old
Figure 3.45 3D Profile of Female (Normal Urine) 70
30-39 years old
Figure 3.46 Pattern for Female (Normal Urine) 71
30-39 years old
Figure 3.47 2D Contour of Female (Normal Urine) 72
40-49 years old
Figure 3.48 3D Profile of Female (Normal Urine) 72
40-49 years old
Figure 3.49 Pattern for Female (Normal Urine) 73
40-49 years old
Figure 3.50 2D Contour of Female (Normal Urine) 74
50-59 years old
Figure 3.51 3D Profile of Female (Normal Urine) 74
50-59 years old
Figure 3.52 Pattern for Female (Normal Urine) 75
50-59 years old
xii
Figure 3.53 2D Contour of Female (Normal Urine) 76
60-69 years old
Figure 3.54 3D Profile of Female (Normal Urine) 76
60-69 years old
Figure 3.55 Pattern for Female (Normal Urine) 77
60-69 years old
Figure 3.56 2D Contour of Female (Normal Urine) 78
70-79 years old
Figure 3.57 3D Profile of Female (Normal Urine) 78
70-79 years old
Figure 3.58 Pattern for Female (Normal Urine) 79
70-79 years old
Figure 3.59 Point plot graph of flux peak (urine glucose) of 80
males and females urine
Figure 3.60 Point plot graph of total flux (urine glucose) of 81
males and females urine
Figure 3.61 2D Contour of Male (Urine Glucose) 84
20-29 years old
Figure 3.62 3D Profile of Male (Urine Glucose) 84
20-29 years old
Figure 3.63 Pattern for Male (Urine Glucose) 85
20-29 years old
Figure 3.64 2D Contour of Male (Urine Glucose) 86
30-39 years old
xiii
Figure 3.65 3D Profile of Male (Urine Glucose) 86
30-39 years old
Figure 3.66 Pattern for Male (Urine Glucose) 87
30-39 years old
Figure 3.67 2D Contour of Male (Urine Glucose) 87
40-49 years old
Figure 3.68 3D Profile of Male (Urine Glucose) 88
40-49 years old
Figure 3.69 Pattern for Male (Urine Glucose) 88
40-49 years old
Figure 3.70 2D Contour of Male (Urine Glucose) 89
50-59 years old
Figure 3.71 3D Profile of Male (Urine Glucose) 89
50-59 years old
Figure 3.72 Pattern for Male (Urine Glucose) 90
50-59 years old
Figure 3.73 2D Contour of Male (Urine Glucose) 90
60-69 years old
Figure 3.74 3D Profile of Male (Urine Glucose) 91
60-69 years old
Figure 3.75 Pattern for Male (Urine Glucose) 91
60-69 years old
Figure 3.76 2D Contour of Male (Urine Glucose) 92
70-79 years old
xiv
Figure 3.77 3D Profile of Male (Urine Glucose) 92
70-79 years old
Figure 3.78 Pattern for Male (Urine Glucose) 93
70-79 years old
Figure 3.79 2D Contour of Female (Urine Glucose) 93
20-29 years old
Figure 3.80 3D Profile of Female (Urine Glucose) 94
20-29 years old
Figure 3.81 Pattern for Female (Urine Glucose) 94
20-29 years old
Figure 3.82 2D Contour of Female (Urine Glucose) 95
30-39 years old
Figure 3.83 3D Profile of Female (Urine Glucose) 95
30-39 years old
Figure 3.84 Pattern for Female (Urine Glucose) 96
30-39 years old
Figure 3.85 2D Contour of Female (Urine Glucose) 96
40-49 years old
Figure 3.86 3D Profile of Female (Urine Glucose) 97
40-49 years old
Figure 3.87 Pattern for Female (Urine Glucose) 97
40-49 years old
Figure 3.88 2D Contour of Female (Urine Glucose) 98
50-59 years old
xv
Figure 3.89 3D Profile of Female (Urine Glucose) 98
50-59 years old
Figure 3.90 Pattern for Female (Urine Glucose) 99
50-59 years old
Figure 3.91 2D Contour of Female (Urine Glucose) 99
60-69 years old
Figure 3.92 3D Profile of Female (Urine Glucose) 100
60-69 years old
Figure 3.93 Pattern for Female (Urine Glucose) 100
60-69 years old
Figure 3.94 2D Contour of Female (Urine Glucose) 101
70-79 years old
Figure 3.95 3D Profile of Female (Urine Glucose) 101
70-79 years old
Figure 3.96 Pattern for Female (Urine Glucose) 102
70-79 years old
Figure 3.97 Point plot graph of flux peak (urine protein) of 103
males and females urine
Figure 3.98 Point plot graph of total flux (urine glucose) of 104
males and females urine
Figure 3.99 2D Contour of Male (Urine Protein) 107
20-29 years old
Figure 3.100 3D Profile of Male (Urine Protein) 107
20-29 years old
xvi
Figure 3.101 Pattern for Male (Urine Protein) 108
20-29 years old
Figure 3.102 2D Contour of Male (Urine Protein) 108
30-39 years old
Figure 3.103 3D Profile of Male (Urine Protein) 109
30-39 years old
Figure 3.104 Pattern for Male (Urine Protein) 109
30-39 years old
Figure 3.105 2D Contour of Male (Urine Protein) 110
40-49 years old
Figure 3.106 3D Profile of Male (Urine Protein) 110
40-49 years old
Figure 3.107 Pattern for Male (Urine Protein) 111
40-49 years old
Figure 3.108 2D Contour of Male (Urine Protein) 111
50-59 years old
Figure 3.109 3D Profile of Male (Urine Protein) 112
50-59 years old
Figure 3.110 Pattern for Male (Urine Protein) 112
50-59 years old
Figure 3.111 2D Contour of Male (Urine Protein) 113
60-69 years old
Figure 3.112 3D Profile of Male (Urine Protein) 113
60-69 years old
xvii
Figure 3.113 Pattern for Male (Urine Protein) 114
60-69 years old
Figure 3.114 2D Contour of Male (Urine Protein) 114
70-79 years old
Figure 3.115 3D Profile of Male (Urine Protein) 115
70-79 years old
Figure 3.116 Pattern for Male (Urine Protein) 115
70-79 years old
Figure 3.117 2D Contour of Female (Urine Protein) 116
20-29 years old
Figure 3.118 3D Profile of Female (Urine Protein) 116
20-29 years old
Figure 3.119 Pattern for Female (Urine Protein) 117
20-29 years old
Figure 3.120 2D Contour of Female (Urine Protein) 117
30-39 years old
Figure 3.121 3D Profile of Female (Urine Protein) 118
30-39 years old
Figure 3.122 Pattern for Female (Urine Protein) 118
30-39 years old
Figure 3.123 2D Contour of Female (Urine Protein) 119
40-49 years old
Figure 3.124 3D Profile of Female (Urine Protein) 119
40-49 years old
xviii
Figure 3.125 Pattern for Female (Urine Protein) 120
40-49 years old
Figure 3.126 2D Contour of Female (Urine Protein) 121
50-59 years old
Figure 3.127 3D Profile of Female (Urine Protein) 121
50-59 years old
Figure 3.128 Pattern for Female (Urine Protein) 122
50-59 years old
Figure 3.129 2D Contour of Female (Urine Protein) 123
60-69 years old
Figure 3.130 3D Profile of Female (Urine Protein) 123
60-69 years old
Figure 3.131 Pattern for Female (Urine Protein) 124
60-69 years old
Figure 3.132 2D Contour of Female (Urine Protein) 124
70-79 years old
Figure 3.133 3D Profile of Female (Urine Protein) 125
70-79 years old
Figure 3.134 Pattern for Female (Urine Protein) 125
70-79 years old
xix
List of Symbols and Abbreviations
Symbol/ Meaning Page
Abbreviation
He Helium 19-106
Ne Neon 19-106
ΕFAS Encircled Flux Analysis System 20-106
W Watt 20-106
LDF Laser Doppler flux 21
2D 2-dimensional 22,66-106
3D 3-dimensional 22,66-106
λ Wavelength 23
SG Specific Gravity 28-106
xx
KAJIAN KORELASI ANTARA PARAMETER URIN DAN PERUBAHAN
FLUKS PADA URIN MANUSIA MENGGUNAKAN LASER He-Ne DAN
SISTEM ANALISIS FLUKS KETERBULATAN
ABSTRAK
Dalam kajian ini, hubungan antara parameter urin dikaji dengan
mendapatkan corak dan ujian statistikal dengan menggunakan SigmaStat 3.1
dan variasi fluks menggunakan laser He-Ne 0.95 mW dan Encircled Flux
Analysis System (EFAS). Data yang diperolehi daripada analisis statistikal
menunjukkan hubungan selari dengan kajian lain. Keselarian ditunjukkan
daripada hubungan yang diperolehi antara pH urin, umur, specific gravity
(SG) urin, urin berprotein dan urin berglukosa. Sebaliknya, keputusan yang
tidak konsisten dengan kajian lain adalah antara SG urin dan umur serta
antara SG urin dan urin berprotein. Kajian parameter urin dengan variasi fluks
mempamerkan corak yang penting. Puncak fluks dan jumlah fluks untuk lelaki
menunjukkan corak peningkatan untuk pH urin dan SG sementara
menunjukkan corak penurunan untuk wanita. Puncak fluks bagi lelaki dan
jumlah fluks bagi perempuan menunjukkan peningkatan dengan umur
sementara jumlah fluks bagi lelaki dan puncak fluks bagi perempuan
menunjukkan penurunan. Corak 2D kontur dan 3D profail memberikan corak
tersendiri berdasarkan jantina, kesihatan urin dan kumpulan umur. Kajian ini
menunjukkan penggunaan laser He-Ne dan EFAS mempamerkan prospek
masa depan yang baik dalam kajian urin. Parameter fluks seperti puncak fluks
xxi
dan jumlah fluks boleh menjadi parameter-parameter yang penting bagi
analisis urin.
xxii
STUDIES ON CORRELATIONS BETWEEN URINE PARAMETERS AND
FLUX VARIATIONS ON HUMAN URINE USING He-Ne LASER AND
ENCIRCLED FLUX ANALYSIS SYSTEM
ABSTRACT
In this research, correlations between urine parameters is studied by
finding patterns and statistical tests using SigmaStat 3.1 and flux variations
using 0.95 mW He-Ne laser and Encircled Flux Analysis System (EFAS).
Data obtained from statistical analysis shows correlations and consistencies
with other researchers. These consistency are reflected from the correlations
obtained between urine pH, age, urine specific gravity, urine protein and urine
glucose. Conversely, the inconsistencies of the results are shown between
urine specific gravity and age and also between urine specific gravity and
urine protein. The studies on urine parameters by flux variations exhibits
significant patterns. Flux peak and total flux for males show increasing pattern
for urine pH and specific gravity while for females show decreasing pattern.
Flux peak for males and total flux for females shows increasing pattern when
aging while total flux for males and flux peak for females shows decreasing
pattern. The pattern of 2D contour and 3D profile gives individual pattern
according to gender, urine health and age groups. This research shows that
using He-Ne laser and EFAS exhibited a good future prospect in urine
research. Flux parameters such as flux peak and total flux, can become
significant parameters for analysis of urine.
3
Chapter 1 : Introduction
1.1 Urine
Urine is a fluid which is continuosly formed in and excreted from the
body. It supplies significant information with regard to many disorders and
diseases [1]. Urine also has been referred to like a mirror, which reflects
activities within the body [2]. It has been identified to presenting a biopsy of the
kidney. It is the principal route of waste removal of products of metabolism from
the body [3]. Disorders of the kidneys obviously modify the composition of the
urine. But kidney disorders may also be complicate many other body processes.
Urine studies may also reflect the situation when the function of the kidney is
normal, but other parts of the body are out of synchronization [4].
The process of urine is formed has been of great interest in science and
medicine [5]. A clear-cut concept of the mechanism of urine formation can be
described, but there are a number of aspects which may be altered or
expanded as additional insight is established. The understanding of mechanism
of formed urine gives a basis for understanding many of the abnormalities of
urine that are observed in disease.
The kidneys are bean-shaped organs and lie retroperitoneally on either
side of the vertebral column. Normally, the 2 kidneys weight about 300 g, and
thus constitute less than 0.5 % of the body weight [6]. The kidneys are quite
close to the abdominal aorta and receive blood through large renal arteries. The
4
cortex or outer portion of the kidney is reddish-brown in colour. This outer layer
of the kidney dips down between adjacent pyramids towards the renal sinus [7].
The basic, microscopic functional unit of the kidney is the nephron.
Figure 1.1 is a schematic diagram of a single nephron [8]. The understanding of
the function of a single nephron provides a basis for understandings of the total
functioning of the kidney. It is estimated that each human kidney contains
approximately 1 to 1.25 million nephrons [9]. The glomerulus lies in the cortex or
outer part of the kidney. The proximal convoluted tubule and the distal
convoluted tubule are situated in the cortex of the kidney, whereas the
descending loop of Henle and the ascending loop of Henle pass almost from
the outer portion of the kidney to the center or medulla and back again. Finally,
the collecting duct passes to the calyx or central portion of the kidney [10].
The afferent arteriole branches into several capillary loops within
Bowman’s capsule [11]. These loops are joined by several anastomoses and
combine to form the efferent arteriole. The proximal convoluted tubule and distal
convoluted tubule are lined with cuboidal cells. The cells are columnar in some
portions of the tubule, quite flat in others [12].
A rich lymphatic network drains the cortex of the kidney, but there is no
significant lymphatic circulation in the medulla or the papilla [13]. The kidney
has an abundant nerve supply which is primarily sympathetic. The nerves have
significant degree terminated in the afferent and the efferent arterioles. The
sympathetic vasomotor nerves are primarily vasoconstrictor in function [14].
5
Figure 1.1. Schematic diagram of a single nephron [8]
The abdominal aorta considers a very great supplying blood to kidneys
through the renal arteries [15]. The arteries subdivide and ultimately become
arterioles which enter the glomeruli in the renal cortex. An ultrafiltration occurs
within the capillary tufts of the glomeruli which are filtered the water and the
solute low weight molecular from the blood [16]. In turn, the blood passes into
the efferent arterioles which are closely approximated to the convoluted tubules.
The formed ultrafiltrate in the glomerulus contents a quite composition of
soluble solutes comparable to the blood which is derived. Therefore the large
molecular weight constituents and cellular elements are both removed. When
the ultrafiltrate passes into the renal tubule various constituents of the
Straight collecting tubule
Loop of Henle
To renal vein
Glomerular capsule
Glomerulus
Branch of renal artery
Afferent arteriole
Efferent arteriole
Proximal convoluted tubule
Distal convoluted tubule
2nd set of capillaries
6
glomerular filtrate are selectively reabsorbed. Therefore, sodium chloride, water,
amino acids, bicarbonate, glucose, uric acid and phosphate are reabsorbed in
the proximal tubule as well as water is reabsorbed in the distal tubule, while in
the collecting tubule, water, sodium chloride and urea are reabsorbed [17]. The
process of reabsorption appears to be delicately regulated by endocrine
mechanisms which involve adrenal cortical hormones and the antidiuretic
hormone.
The proximal tubule absorbs large amounts of glucose, the fluid passing
through this segment of the nephron retains the same osmolality as plasma
[18]. The development on concentrated urines with high osmotic pressures is
achieved due to the changing in osmolality in the distal tubule. Alternatively, the
most of urine may lose its electrolytes and become quite dilute in this portion of
the kidney. In the tubules, there is also an active process of tubular excretion
which involves the excretion of numerous substances from the blood directly
into the tubular urine [19]. The hydrogen ion is one of the substances excreted
by the tubular cells, which promptly combines with ammonia or phosphate.
In order of magnitude, one might envision that there are approximately
1,000 litres of blood pass through the kidneys each day and approximately 100
litres of glomerular filtrate are formed during this period [20]. Then, most of this
filtrate is reabsorbed, at that a final typical urine volume is 1 litre/day. From a
functional standpoint, the process of urine formation provides the excretion of
waste products and the regulation of body water, body pH, and body
electrolytes [21].
7
Urine can be considered the most complex fluid in the body. It contains
practically all of the constituents found in the blood. Although many substances
found in the blood and in the urine, the concentration are different in the two
body fluids. In many instances, the amount of a given urinary substance may far
exceed the amount present in the blood. For example, urea has concentration
with a normal blood about 20 mg/100 ml and with a normal urine about 3,000
mg/100 ml [22]. For other substances, the concentration present in urine may
be very much less than in blood. Glucose is one of this type of substance,
where the fasting blood concentration is about 90 mg/100 ml and the urine
concentration is closer to 10 mg/100 ml [23].
The urine is a sparklingly clear fluid generally, which is yellow or amber
[24]. It has a characteristic’s odour which is not regarded as disagreeable by
most persons. The urine may be turbid and yet be completely normal. Turbidity
of urine specimens in healthy persons is due to precipitation of phosphate salts
or uric acid in the bladder [25]. Such precipitation may occur due to changes in
the acidity or alkalinity of the urine in the bladder. The odor of urine have
modified in order to the kind of foods in the diet [26].
One of the essential functions of the kidney is excreted a waste materials
and substances, which the body are not needed. Urine density is related
primarily to an amount of excreted water, salt and urea [27]. The osmolality of
the urine or other body fluids is an expression of the osmotic pressure.
Osmolality and urine density are quite closely related, with the advantage that
8
expressing values in osmolal units permits comparison of urine with blood and
thus provides a slightly greater convenience in identifying renal activity [28].
Most of circumstances of human urine, the specific gravity is between
1.008 and 1.030, but the ingestion of large amounts of fluids decreases the
specific gravity almost to 1.000 [29]. Someone does not ordinarily find the
significant specific gravity in excess to 1.030, unless metabolites of certain
drugs are being excreted or a large quantity of glucose or protein is existed. The
ability of the kidney for excretion a dilute or concentrated urine is frequently
measured by a dilution-concentration test [30]. Various procedures are used in
such studies, and all are define how the kidney responds to a condition and
situation wherever is need to excrete an increase water and deprivation of mild
fluid [31].
Urine pH measurement is a part of most regular urinalysis [32]. The pH of
urine is affected to a significance degree by the acidic or basic salts which are
in the specimen. The mechanism of excretion acid or alkaline urine that the
body can get rid of relatively large amounts of either acids and/or bases and
maintain a constant homeostatic state [33]. Some chemical constituents of urine
are mainly responsible for establishing the pH of any specific urine specimen
[34]. These substances included sodium and potassium mono- and dihydrogen
phosphates, sodium citrate, ammonium salts, sodium bicarbonate and carbonic
acid. A great number of other substances have been normally made a smaller
contribution to the final urinary pH. The majority of the substances are simply
excreted from the blood into the urine by the kidney [35].
9
However, in the case of ammonium salts, the kidney actually converts a
neutral urea into ammonia, providing a mechanism to excretion of acids from
the body [36]. This conversion process is quite active in situations where the
body tends to have an excess of acid. Correspondingly, if the body has an
excess of base, the kidney synthesizes citrate in relatively large quantities, thus
providing a mechanism for excretion of extra base [37]. Table 1.1 shows a
comparison of the pH of urine, various body fluids and other material .
The pH of the urine of a healthy person reflects the acid-ash or alkaline-
ash composition of the diet [38]. During the course of a day, the urine pH will
ordinarily show rather rapid and large swings from acid to alkaline or vice versa.
This can be recognized by a specimen being turbid at the voided time. This
turbidity is most frequently caused by the fact that certain components which
are quite soluble in an acid urine are precipitated when the specimen is made
alkaline, as by the admixture in the bladder of an excess of alkaline urine [39].
Alternatively, certain substance that are soluble in an alkaline urine will
precipitate if an excess acidity is established.
10
Table 1.1. The pH of urine compared with body fluids and other material [40]
The processing of adjustment of urinary pH by the kidney occurs in both
the proximal tubule and the distal tubule, where a selective absorption of
bicarbonate or secretion of ammonia occurs. According to current concepts of
the process of urine formation, the glomerular filtrate has a pH, which is
essentially the same for the blood. As the urine proceeds along the proximal
tubule, the pH is lowered to 6.8 [41]. This occurs primarily as a result of
selective reabsorption and tubular excretion. When a decrease in pH takes
place, the active secretion of hydrogen ion occurs in the distal tubule and the
pH may drop to values of less than 5. Ammonia is secreted in the distal tubule
and due to the exist acid, it promptly combines to form an ammonium complex
which is excreted in the urine.
Body fluids and other material pH Urine 4.8 – 8.5 Blood 7.4 Serum 7.4 Plasma 7.4 Saliva 6.75
Gastric juice 1.2 – 3.0 Pancreatic juice 8.7
Bile 7.5 Duodenal fluid 6.7 Jejunal fluid 6.5
Ileal fluid 7.1 Aqueous humor 7.2
Sweat 5.2 Milk 7.0
Semen 7.4 Tears 7.4
Interstitial fluid 7.4 Intracellular fluid – liver 6.9
Sea water 7.3 Tomato juice 4.3
Grapefruit juice 3.2 Cola soft drink 2.8 Lemon juice 2.3
11
During acidosis, ammonium excretion increases from 20 - 30 meq/day to
more than 500 meq/day [42]. The cells of the tubule generate ammonia from a
variety of amino acids, most notably glutamine [43]. The kidney loses its
capacity to generate a significant degree of ammonia. Thus, the renal
impairment patients lose their ability to excrete an acid load maximally. This
tends to cause an acidosis in such patients. Addison’s disease have also an
impaired ammonia forming mechanism, which promptly disappears when
corticosteroids are administered [44].
The extreme range of urine pH is change from pH 4.8 to pH 8.5
approximately [45]. In situations of extreme ketosis, the urine have a slightly
lower pH, and in instances of severe infections of the kidney or bladder, the pH
of the excretion urine excesses of pH 9 due to alkaline ammonium carbobate
which is formed from urea. The physiological capability of tubular cells to
selectively respond to very slight changes within the body (changes so slight
they cannot at present be measured by available instrumentation) represents a
most highly refined biological regulating mechanism [46].
At least three genetic disorders relate to loss of the ability of the distal
tubular cells to make their contribution to body pH control [47]. In renal tubular
acidosis, the kidney is incapable of forming a highly acid urine and accordingly
when an excess of acid is presented to the body, the kidney is not able to
contribute its usual control function. Therefore, the urine remains about neutral
and severe acidosis results. In renal tubular alkalosis, tubular cells are unable to
excrete an alkaline urine so that when alkali excesses are presented, alkolisis
12
ensues. In the Fanconisyndrome, loss of renal acid excretory ability occurs with
resulting acidosis [48].
If a large amount of water is ingested by a human, a corresponding
diuresis or increase in urine excretion occurs. At this time, the pH of the urine
tends to become relatively fixed at a value quite close to neutrality [49]. This
phenomenon is indicated that the normal process of urine pH adjustment in the
proximal and distal tubules does not have an opportunity to function effectively.
The pH of the urine becomes quite close to the blood. Quite comparable effects
on pH are seen when mannitol administration have made diuresis to occur in
either humans or experimental animals [50].
Tests for glucose in urine have done more frequently than any other
single chemical or biological urinary measurement [51]. Such tests have done
with great frequency in the procedures of screening healthy person for the
identification of asymptomatic disease, as a part of diagnostic workup in the
recognition of diabetes, or for differential diagnosis in resolving the problems of
the crises of diabetes. Eventually, tests of sugar in the urine provide an
important monitoring mechanism for diabetic patients to assess the
effectiveness of their control by medication or by diet [52]. This gleaning
information of the diabetic is utilized by the physician in the regulation of the
disease.
Within recent years, there has been marked an increment in the use of
urine sugar tests which reflects the expansion on screening tests and patient
13
monitoring of the treatment of their disorder. It is frequently known that normal
urine does not have glucose, but need to qualify since there is a minute quantity
of glucose in all normal urine [53]. However, both specific tests of glucose which
employ enzymes and non-specific tests for reducing sugar have adjusted
sensitivity so the normal urine gives a negative reaction [54].
The concentration of glucose in the blood is usually between 65 and 80
mg / 100 ml during the fasting time [55]. This concentration does not decrease
according to the mechanism of the formation of glomerular. The rate of
reabsorbtion is actually a little lower in glucose than in water. It means that the
final urine contains a very minute amount of glucose. Schersten and and Fritz
[56] have indicated that normal urine usually contains more than 2 mg / 100 ml
than up to 20 mg / 100 ml of glucose concentration.
The concentration of glucose in the glomerular filtrate is increased
correspondingly when the blood sugar is concentrated on more than 180 – 200
mg [57]. Instancelly, the relative amount of water reabsorption greatly exceeds
the amount of glucose reabsorption and the capability of the tubule cells to
phosphorylate so that lead to increase a glucose concentration in the urine
which is ranging from 100 mg / 100 ml to more than 10,000 mg / 100 ml [58].
Above of this level is known as the renal threshold.
The kidney is reabsorbed these kinds of sugars, galactose, fructose,
xylose, lactose, sucrose, mannose and others in the same way of glucose [59].
These sugars will appear in the glomerular filtrate, when they are exist in the
14
blood and will not be as rapidly reabsorbed as the water of the filtrate.
Accordingly, there is a quantity very much greater than in blood in the final
urine. No more sugars rather than glucose appear to have a renal threshold
[60].
A test for the presence or absence of protein in the urine is one of the
most frequently performed procedures in routine urinalysis [61]. Before more
than one century, clinical tests were based on precipitation phenomena
involving the coagulation of protein by heat and various chemical agents,
including concentrated nitric acid, trichloroaceetic acid and sulfosalicylic acid
[62]. The Merck Index, 5th edition [63] listed about 33 different tests for protein
(albumin) in urine. In 1957, the dip-and-read colorimetric test in most parts of
the world [64]. The colorimetric dip-and-read test provides a satisfactory degree
of specificity and also gives a semiquantitation of the exist amount of protein.
There are also tests which identify the presence of various specific proteins or
groups of protein in the urine is a screening procedure which is applied to all
patients and utilized for evaluation studies with healthy subjects [65].
The mechanism of protein to be into urine is not completely obvious.
Kark et al. [66] suggested five possibilities pathways of entry of protein into
urine that were passage of protein across the glomerular membrane,
disturbance of the normal tubular resorption of protein, abnormal secretion of
protein from the plasma by the tubular cells, loss of plasma proteins from the
lymphatics of renal papillae and abnormal secretion of genitourinary tract
proteins.
15
More than one of these mechanism might be explained the excretion of
protein in urine. Rennie [67] has reviewed and discussed the subject of
proteinuria and has indicated that the evidence is quite overwhelming in support
of the concept that protein leaks from the serum through the glomerulus and
subsequently reabsorbed in the tubule. Ordinarily, there is a clear-cut relation
between the size of the protein molecule and its rate of clearance.
Thus, by all the mechanism and physical properties of urine, it is
obviously an amazingly complex entity which has many advantages to study.
Urine has much information of a varied nature to contribute the measurement of
many chemical and physical parameters. The analysis of urine can provide
important information of the body functions and health.
16
1.2 Helium-Neon Laser
The laser tube in a He-Ne laser consists of a long a discharge tube filled
with the active medium that is a mixture of about 10 parts of helium to one part
of neon [68]. The gas mixture of helium and neon forms the lasing medium and
this mixture is enclosed between a set of mirrors forming a resonant cavity
consists of a plane, high-reflecting mirror at one end of the laser tube, and a
concave output coupler mirror of approximately 1% transmission at the other
end.
The red He-Ne laser wavelength is usually reported as 632.816 nm [69].
This is in fact the wavelength in air, and corresponds to a vacuum wavelength
of 632.991 nm. The precise operating wavelength lies within about 0.002 nm of
this value, and fluctuates within this range due to thermal expansion of the
cavity [70]. The light emission from gas lasers as compared to that from solid
state lasers is found to be more directional and much more monochromatic [71].
This is due to the various imperfections present in the solids and also the
heating caused by the flash lamp. Even though solid state diode lasers can now
provide red laser light beams with intensities comparable to those obtained with
He-Ne lasers, it is anticipated that the He-Ne laser will remain a common
component in scientific and technical instrumentation in the foreseeable future
[72].
In this research, 0.95 mW He-Ne laser of wavelength 632.8 nm is used to
find the correlations of urine parameters and flux variations of human urine.
Significant relationships between urine parameters is studied by obtaining
patterns and statistically tests using SigmaStat 3.1. Urine parameters have
17
Helium + Neon Laser beam
Mirror Mirror
Discharge electrodes
studied on flux variations using He-Ne laser and Encircled Flux Analysis System
(EFAS). The functions of He-Ne laser in medical fields can be wider by using it
as a tool to study the changes that happened in the urine in corporation with
EFAS. It can contribute to medical investigation for diagnosis or theraphy
planning. This thesis study and discuss the flux peaks and total flux patterns of
males and females in the age groups ranged 20-29 years to 70-79 years old.
.
Figure 1.2. Schematic of helium-neon laser [73].
1.2.1 Flux of Laser
. The flux of a quantity is defined as the rate of flow of energy through a
given surface [74]. Flux has been used in laser ablation which is the process of
removing material from a solid (or occasionally liquid) surface by irradiating it
with a laser beam [75]. It also been used to study a characteristic pattern of
18
laser Doppler flux (LDF) of patients with venous ulcer before and after ulcer
healing [76]. The flux became a significant parameter in the study on laser
needle acupuncture [77]. The changes in human urine when aging also has
been studied by finding patterns and correlations in flux variations [78]. In this
research, the flux peak and total flux are been used as a significant parameters.
The flux peak is the value of the peak flux in counts while the total flux is the
value of the total flux in the beam area.
1.3 Objectives of the Research
It is most likely that one of the trends of the future will be to utilize urine as
a means of reinforcing the concept that a state of health prevails [79]. The
correlation of more information of the urine certainly must be effectively utilized.
There also a significant progress in the evolution of new as well as improved
chemical and physical test systems which are important in urine study.
Hence, the focus of this research are to find out the correlations between
urine parameters such as urine pH, urine specific gravity, urine protein, urine
glucose and age. This research also aim to obtain the range of flux levels of
patients urines and to observe the 2D contour and 3D profile of the patients
according to gender.
19
1.4 Outline of Thesis
As an introduction to the background theory, Chapter 1 contains a
summary of human urine and Helium-Neon laser which are the fundamental
knowledges in this study. In Chapter 2, attention is paid to the explanation of the
urine samples, instrumentation and experimental set-up used in this research.
Chapter 3 discusses the obtained results and statistical analysis techniques
used to study the samples whereas Chapter 4 contains the 2D countour and 3D
profile images. Finally, Chapter 5 summarises the conclusions of this thesis,
and recommendations for future work.
1.5 Literature Review
This research used a new technique which not being done yet by other
researchers. Because of that, there were no published journals or references
that have used laser in their urine research. There were some researchers that
used the conventional methods to study urine test and correlation with disease
such as about urinary tract infection [80]. Consequently, urine is widely studied
as an aid in diagnosis and monitoring the course of treatment of disease [81].
The correlation between urine parameters has been studied by other researcher
such as P. Tabouleta et al [82] that doing research on the correlation between
urine ketones (acetoacetate) and capillary blood ketones (3-beta-
hydroxybutyrate) in hyperglycaemic patients . There are also study on
correlation between urine and the patients that under the influence of drugs of
abuse [83] .
20
In the medical applications, He-Ne laser has been widely used in various
thrust. It is expected to be used extensively in the treatment of cancer [84]. He-
Ne laser has been used for clinical PhotoDynamic Therapy in China [85].
Helium-Neon laser also has became therapeutic tool to reduce the risk of acute
myocardial infarction in the patients [86] and the treatment of diabetic patients
[87].
There are two main methods to do the urine test in the hospitals and
clinics in Malaysia, there are by using reagent dipstick which will change colour
when in contacts with urine and laboratory test which will use machine such as
Urisys 1800 that being used in Penang Hospital. The using of this laser
technique to do the urine test is better than other conventional techniques
because of more faster, cleaner and easier procedures. This technique is also a
better technique because it can give the reading in flux parameters such as flux
peak which is the peak counts in the beam area and total flux which is the
amount of flux in the beam area.
21
Chapter 2 : Materials And Methods
2.1 Urine samples
Human urines were kindly provided by Penang Hospital, Malaysia. A total
of 160 urine samples from 82 males and 78 females were used in this research
(Figure 2.1). Urine samples were obtained from patients range from 20 to 79
years old. 5 plastic containers of urine were obtained in 5 days every week
from Monday to Friday. The urine were received at 9.00 am in the morning and
by 11.00 am, the process of labelling were done to the urine samples in the lab.
The samples were labelled with the coding systems according to gender,
disease and age.
2.2 Instrumentation
The 0.95 mW He-Ne laser has been used as a power source and
Encircled Flux Analysis System (EFAS) Model 8350, Photon Inc. as a flux
detector (Figure 2.2). He-Ne laser (λ=632.8 nm) has been choosen as a power
source because of its properties operation which are suitable for this research
work and non-destruction tests can be done that it is very important for the
samples. The major system hardware components were Photon Model 2320
BeamProfiler CCD camera with built-in variable optical attenuator, 60X objective
lens, Photon Magnifying Objective Lens Mount for mounting the camera and
lens, high precision XYZ translation stage with rail mount, Photon Model 3180
22
Controller, Photon BeamProfiler Image Capture Card and Encircled Flux Analysis
Software for Windows 95/98/ME and Windows NT4.0/2000 Professional. A
statistical software named SigmaStat 3.1 has been used to perform effective
and accurate statistical analysis and interpretation to the results.
2.3 Experimental methods
The research was carried out in the lab after the proccess of labelling
and coding have been done to the samples. A 5 ml catridge was used to draw
the urine from the plastic container and 0.2 ml drops of each urine sample was
injected on 7.5 cm x 2.5 cm slide. The slide was placed between power source
and the detector, 12 cm away from He-Ne laser and 1 cm from EFAS which was
connected to the computer that installed with its software (Figure 2.3). Then, the
He-Ne laser was switched on and a set of 10 readings were obtained for each
urine sample. In order to avoid interference from the environment light, the
whole set-up was placed in a box which acts as a research platform (Figure
2.4). After the study have been completely done, all of the urine samples in the
plastic container was discarded in the black plastic into the dustbin.
The values of the flux peak and total flux and also the 2D contour and 3D
profile will be show on the computer screen. The graph are plotted from the
obtained values. The values also been analized statistically using SigmaStat
3.1. To obvious more clearly the patterns of 2D contour and 3D profile, the
freeform line is used by joining the highest beam intensity on the beam area.
23
Figure 2.1. Urine samples
Figure 2.2. He-Ne laser (0.95 mW) and Encircled Flux Analysis System
(EFAS) Model 8350, Photon Inc.
Encircled Flux Analysis System
He-Ne laser
24
Figure 2.3. Schematic diagram of the experimental set-up.
Figure 2.4. The box that was used to avoid interference from the environment
light and acts a research platform.
1 cm
He-Ne laser Encircled Flux Analysis System
12 cm
Slide
Urine
Encircled Flux Analysis System
Computer
Wire