Post on 30-Sep-2020
56
Results
57
Results
The present study was started at the Nellore district, Andhra
Pradesh, India. The Udayagiri mandal of Nellore district seems to be
more threaten area of fluoride toxicity in drinking water. A sum of total
ten fluoride affected villages have been find out with the help of water
control department and the water samples were collected for the
analysis of fluoride content. Water samples from different bore wells of
ten villages showed a maximum range of 2.37 to 6.74 ppm (Table-3;
Fig. 5) by SPADNS method. Among the selected ten villages three have
shown high levels of fluoride content in their drinking water (range 4-7
ppm). Particularly Varikunta padu showing a maximum fluoride content
of 6.74 ppm. In addition to that total water analysis was made in the
water samples from the ten villages. This analysis includes the pH,
alkalinity, hardness, Ammonia, Calcium and magnesium in addition to
fluoride content (Table-4). All these parameters have shown
abnormality in all the selected areas. These three villages namely,
Varikunta padu (6.74 ppm), Kolangadi palli (5.12 ppm) and Gangireddy
palli (4.43 ppm) were taken for the further entire study.
Analysis of the samples showed the fluoride content in abnormal
range both in urine and serum (Table-5; Fig. 6). The generally accepted
average normal serum fluoride value is 8 µM (0.15 ppm.) as found by
Singer and Armstrong (1977). Incase of urine fluoride acceptable point
58
is 1 mg per liter. But in the case of the selected objects it seems to be
more when compared to the normal value. Particularly Kolangadi palli
people showed a maximum of 2.27 ppm of serum fluoride and in case
of urine fluoride Varikuntapadu people showed a maximum range of
4.00 mg. With this background further analysis has been made in the
selected three specific villages with strength of 90 subjects.
A detailed data has been collected from the villagers of the
selected areas which include the personal details like age, sex, duration
of stay in the specified area, drinking water source, and parental history
and present or previous experience of diseases like diabetes and
hypertension. It helped us to omit the people suffering with hypertension
or diabetes, where there is a chance of getting the renal failures with the
hypertension or diabetes. After screening of the data we have selected
a sum of 90 people, who are never suffered with hypertension or
diabetes.
After selecting the villagers their urine and serum samples were
collected for the analysis of fluoride content. While collecting the
samples we have noticed their details like height, weight, waist and hip
ratio and other details. Even though the subject selection was done
specifically , again a cross check has been made to know the random
blood glucose levels as well as blood pressure of the selected 90
fluoride threaten individuals (Table-6; Figs.7,13). Blood pressure was
59
measured with the help of a local rural medical practitioner by using
Sphygmomanometer. These results showed that the difference was not
significant (p<0.001) and there was not much change when compared
to that of control value. Mean value of RBS showed to be 175 mg/dl,
where as control mean value is 173 mg/dl (Table-6).
Measuring serum creatinine is a useful and inexpensive method
of evaluating renal dysfunction. Creatinine is a non-protein waste
product of creatine phosphate metabolism by skeletal muscle tissue.
Creatinine production is continuous and is proportional to muscle mass.
Creatinine is freely filtered and therefore the serum creatinine level
depends on the Glomerular Filtration Rate (GFR). Renal dysfunction
diminishes the ability to filter creatinine and the serum creatinine rises.
If the serum creatinine level doubles, the GFR is considered to
have been halved. A threefold increase is considered to reflect a 75%
loss of kidney function. Present study revealed that there was a drastic
increase, almost doubled with the control value indicates the loss of
renal function (Table 6; Fig. 8). Control subjects showed the creatinine
content of 1.43 mg/dl, where as the disordered subjects showed a value
of 2.78 mg/dl, which shows a drastic increase in the serum creatinine
value and the loss of renal function. From the results it can be observed
a significant (p<0.001) increase in the serum creatinine content.
60
After serum creatinine evaluation and confirmation of the selected
subjects were exempted from diabetic and hypertension people, the
studies were further extended to know the alterations at different levels.
Complete blood picture can provide a clear picture of the cellular as well
as chemical components of the living system. Hence, studies were
conducted in the control as well fluoride affected people. Selected
objects blood samples were used for the analysis. Table-7 shows some
of the important hematological parameters like WBC, RBC, Hb
percentage, HCT, MCV and MCH. Results showed that there was a
slight increase in WBC content, shows a significant (p<0.001) increase.
But in the case of other parameters there was a different response.
Except WBC reaming all mentioned parameters showed to be
significantly (P<0.001) decreased. In the case of HCT and MCV there
was a much difference when compared to that of controls. In case of
controls HCT and MCV showed 41.92 and 92.16 fL respectively. But the
values decreased to 38.46 and 81.97fL respectively in selected patients
(Table-7; Fig. 9).
Table-8 shows the continuation of hematological parameters like
MCHC, PLT, RDW-SD, RDW-CV, PPW and MPV. With these
parameters also there exists significant (p<0.001) increase in the case
of MCHC and PLT i.e. 28.86% and 151.47 mm3 in controls, where
61
affected subjects showed 30.85% and 198.22 respectively. Remaining
all parameters were shown to be decreased (Table-8; Fig. 11).
Table-9 shows the continuation of parameters of haematology like
P-LCR, LYM percentage, MXD percentage, neutrophils, lymphocytes
number, MXD number and NEUT number. Here also it was found
increase in some of the parameters like LYM percentage, MXD
percentage, lymphocytes number, MXD number and NEUT number.
Particularly drastic increase was observed in MXD percentage i.e. 2.57
in control where as fluoride affected persons showed 8.58, which shows
a significant (P<0.001) increase. Next to this there was a significant
(P<0.001) increase was noted in case of LYM percentage. Control value
shows 38.62 whereas fluoride threatens subjects showed 44.26 (Table
9; Fig. 10).
After the determination of changes in the complete blood picture,
studies were conducted to know the next important parameters i.e. lipid
profile. Any change in the body will reflect immediately in lipid content of
the biological system. Particularly alterations in any metabolic activity
reflect in the altered lipoprotein content as well as in the cholesterol.
Here in the case of fluoride toxicity also we have conducted experiment
to know the lipid profile of the fluoride toxic subjects. From the results it
was clear that there was a drastic enhancement in all lipid parameters
except in HDL (Table 10; Fig. 12). In case of triglycerides control
62
subjects showed 122.72 mg/dL, where as the fluoride altered patients
showed 146.96 mg/dL which shows a drastic increase, which shows a
direct relation between the increased fluoride concentrations and the
lipid metabolism and accumulation of fat content in the blood stream.
For cholesterol control people shows 147.4 mg/dL, where as test
subjects showed an increase to 164.09 mg/dL. In case of VLDL and
LDL also there was an increase when compared to that of controls
(Table-10). Figure 12 shows a comparative altered lipid profile in the
control as well as treated subjects. But they are not seems to be higher
as in the case of heart disorders. This gives an idea that fluoride is not
much involved with lipid metabolism comparatively.
As the lipid profile completely describes about the cardiac
function and abnormalities of lipid metabolism in the renal failure
patients, next delicate and foremost important organ in the human body
is liver. Any abnormalities in the liver due to the toxicants will develop
the free radical mediated damage, where the detoxification can be done
at liver it self. Hence we have gone for the analysis of liver function
under fluoride toxicity.
The commonly used liver function tests (LFTs) primarily assess
liver injury rather than hepatic function. Indeed, these blood tests may
reflect problems arising outside the liver, such as hemolysis (elevated
bilirubin level) or bone disease (elevated alkaline phosphatase [ALP]
63
level). Abnormal LFTs often, but not always, indicate that something is
wrong with the liver, and they can provide clues to the nature of the
problem. However, normal LFTs do not always mean that the liver is
normal. Patients with cirrhosis can have normal LFTs. Abnormalities in
LFTs are elevated levels of static biochemical tests, including aspartate
aminotransferase (AST) (formerly serum glutamic-oxaloacetic
transaminase [SGOT]), alanine aminotransferase (ALT) (formerly serum
glutamate pyruvate transaminase [SGPT]), alkaline phosphatase,
bilirubin, albumin, globulin and A/G ratio.
Table-11 explains about the detailed analysis of LFTs, where we
can find a minute enhancement with the control values. But there was
no one parameter showing drastic change. Al most all parameters are in
normal range of reference values. SGOT and SGPT enzyme activities
were found to be slightly decreased when compared to control values
(Figure-15). Serum bilirubin was also in normal range both in control
and treated subjects. Protein content incases of albumin and globulin
seems to be slightly increased (Table 11 and Figure-16). This indicates
fluoride is not showing much toxicity in the liver and even enzymatic
activities were also seems to be not much elevated. Thus LFT results
indicated that there was not much significant (P<0.001) alteration in the
fluoride affected people.
64
As fluorine is the most electronegative element, distributed
ubiquitously as fluorides in nature and its involvement with biological
system through water it defiantly alters the electrolytes in the biological
system. Sodium and potassium are the two important electrolytes which
maintain the homeostasis, acid base balance and also different
biological functions in the membranes. Hence further studies were
made to know the levels of two important electrolytes (sodium and
potassium) in the serum of the control as well as the fluoride threaten
people. Table-12 shows the analysis of serum sodium and potassium
levels of the selected subjects. Sodium levels shows to be increased in
the selected subjects. Control value of serum sodium is 134.81mmol/L
and in case of the treated persons showing 139.37mmol/L, that
indicates fluoride increases significantly (P<0.001) the sodium
concentration in the serum (Table-12; Fig. 17). In case of potassium it
was showing similarity to that of sodium. The control value was 4.04
whereas fluoride affected people showing a slight significant (P<0.001)
increase of 4.64. This clearly suggests that fluorine is involved with the
electrolytes and altering the sodium and potassium levels.
The routine classical evaluation of nephropathy (any type of renal
problems) includes the identification of glomerular and tubular markers
in the patient’s serum and urine. The normal individual doesn’t contain
this content elevated in their urine or in serum samples. These
65
glomerular and tubular markers include: transferrin, Ig G, antitrypsin, β-
2-microglobulin and angiotensin converting enzyme 1 (ACE-1). Recent
studies also have demonstrated that, there were tubular components in
renal complications of disease conditions as shown by the detection of
renal tubular enzymes and low molecular weight proteins in the urine as
well as in serum. In fact, tubular involvement may precede glomerular
involvement because several of these tubular proteins and enzymes are
detectable even before the appearance of micro albuminuria and rise in
serum creatinine (Catalano et al., 1993).
Thus studies were conducted to evaluate the glomerular and
tubular marker in urine as well as in serum of the control and fluoride
affected people. Table-13 shows the analysis of serum glomerular and
tubular markers in the control and test samples. Transferrin is a plasma
protein that transports iron through the blood to the liver, spleen and
bone marrow. The blood transferrin level is tested for diverse reasons
like to determine the cause of anemia, to examine iron metabolism (for
example, in iron deficiency anemia) and to determine the iron-carrying
capacity of the blood. Low transferrin can impair hemoglobin production
(since to make hemoglobin, you have to have iron) and so lead to
anemia. Low transferrin can be due to poor production of transferrin by
the liver (where it was made) or excessive loss of transferrin through the
kidneys into the urine. Here in the present study the level of transferrin
66
seems to be low when compared to that of control (Table-13). This
indicates the chance of anemia due to fluoride toxicity. Low levels of
IgG occur in macroglobulinemia. In this disease, the high levels of IgM
antibodies suppress the growth of cells that produce IgG. Other
conditions that can result in low levels of IgG include some types of
leukemia and a type of kidney damage (nephrotic syndrome). Here we
can find the low levels of serum IgG, but with in the normal range
indicating the altered renal function (Table-13; Fig. 18).
Alpha 1-antitrypsin (A1AT) is produced in the liver. Accumulation
of this in liver causes lower levels of A1AT in blood results in the
development of liver cirrhosis. Excessive excretion of A1AT through
urine indicates the loss of renal function. In present case there is no
difference with the control value. It seems to be almost equal, that
indicates the normal functioning of liver (Table-13). Beta 2-
microglobulin is a protein found on the surface of many cells. Testing is
done primarily when evaluating a person for certain kinds of cancer
affecting white blood cells including chronic lymphocytic leukemia, non-
Hodgkin's lymphoma, and multiple myeloma or kidney disease. In our
study it was very interestingly rapid enhancement of B2M was noticed.
The control subjects showed 3.03 mg/L, where the fluoride
affected people showed a maximum increase of B2M to 10.60 mg/L.
67
This shows a drastic increase which indicates the altered renal activity
in the fluoride affected people (Table-13). There was a significant
(P<0.001) increase compared to the normal. This altered range is more
supportive for further analysis for the fluoride mediated renal damage.
The angiotensin-converting enzyme test is used to measure the blood
level of angiotensin-converting enzyme, which converts angiotensin I to
angiotensin II and controls blood pressure. Angiotensin-converting
enzyme and ACE2 are highly expressed in the kidney. The role of ACE
in the development of renal damage is generally accepted. In the
present study the ACE level seems to be decreased when compared to
that of control individuals (Table-13; Fig. 19). Control individuals having
a concentration of 44.97 U/L, and fluoride affected people are showing
a concentration of 37.07U/L, indicating a significant (P<0.001)
decrease.
After identifying the glomerular and tubular markers in the serum
studies has been made to know the status of the same in the urine to
confirm the fluoride mediated renal failures. From the table-14 and
fig.20 it is clear that transferrin levels are hiked in the fluoride affected
people. Control people are showing a value of 195.50mg/dl and the
fluoride affected are showing 221.43 mg/dl. From the earlier table it
was clear that transferrin concentration seems to be low in the serum
and now it increases in the urine indicates the loss of renal function
68
(Table-13 and 14). Similar results were found in the case of IgG in the
urine as well as in serum. Here also it was found the decreased
concentration of serum IgG and increased levels of urinary IgG
indicating the renal alterations. The control urinary IgG seems to be
34.54mg/dl and in the threaten people it reaches to 45.41mg/dl. This
shows a significant (P<0.001) increase (Table-14; Fig. 20). But in the
case of A1AT it was changed, where the serum A1AT is not having any
significant change. But here it was clearly found the altered values of
A1AT.
The control individuals were showed 3.61g/L A1AT, where the
affected people have shown an increased value of 39.96g/L indicates
the increase in the excretion of A1AT due to renal failure (Table-14).
B2M also showing similar pattern of over excretion. Here we can find
3.64mg/L in the treated people where the control value is 1.24mg/L. It
was found to be drastically increased in the serum as well as in urine of
the affected people. The same was also found with ACE levels where
the control value was 11.46U/L and the treated people were showing
13.77U/L, which means over excretion indicates the renal problems
(Table-14).
An increased level of B2MG as well as ACE indicates the kidney
failure. But to know the actual mechanism further studies have been
69
made. Genetic predisposition studies suggest a potential role of genetic
factors in the pathogenesis of renal failures at any cause and the gene
encoding angiotensin-I converting enzyme (ACE) is a potential
candidate gene in its etiology. ACE, a potent vasoconstrictor, catalyzes
the conversion of angiotensin I to angiotensin II. It also inactivates
bradykinin, a vasodilator, by bringing about its proteolysis (Crisan and
Carr, 2000). Hence present study was designed to study the association
of ACE polymorphism to the attribution of fluoride mediated renal
damage.
The DNA samples from 90 fluoride mediated nephropathy and 60
normal healthy controls were amplified for I/D polymorphism in the ACE
gene and analyzed. Figure-21 represents the PCR products of 190 and
490 bp indicating the presence of deletion (DD) and insertion (II)
genotype respectively. The preferential amplification of the D allele and
inefficiency of the amplification of I allele may result in the mistyping of
ID heterozygotes as DD homozygotes. Therefore, in order to increase
the specificity of DD genotyping, all samples, identified as DD after
initial amplification were reconfirmed with an insertion-specific primer
pair, as mentioned in material and method section. The presence of
insertion sequence was revealed by the amplification of a 275 bp
fragment, while DD homozygote failed to amplify due to the lack of
annealing site (Fig. 22).
70
Table-15 shows the distribution of ACE genotypes in fluoride
mediated nephropathy patients and normal controls. The frequency of D
allele and DD genotype was only marginally higher in fluoride affected
patients as compared to the normal controls. The observed and
expected genotypic frequencies were in Hardy-Weinberg Equilibrium.
71
Table 3: Fluoride contents in water samples of the selected tenVillages in and around Udayagiri mandal (Nellore district,A.P., India)
Name of the
village
Fluoride content in
water in ppm
Turkapalli 4.01
Pakeerpalem 4.00
Varikunta padu 6.74
Bijjam palli 2.92
Masi peta 2.37
Singa reddy palli 2.98
Boda banda 3.47
Kolangadi palli 5.12
Gangireddy palli 4.43
Basine palli 3.12
72
Table 4: Complete drinking water analysis of the selected areas inand around Udayagiri mandal (Nellore district, A.P., India)
Name of the
village
pH Alkalinity
ppm
Calcium
ppm
Magnesium
ppm
Hardness
ppm
Ammonia
Ppm
Turkapalli 7.45 620 25 365 390 0.02
Pakeerpalem 7.50 645 35 315 350 0.01
Varikunta
padu
7.90 655 30 405 435 0.06
Bijjam palli 7.63 630 24 406 430 0.03
Masi peta 7.43 545 32 378 410 0.01
Singa reddy
palli
7.81 440 26 334 360 0.00
Boda banda 7.21 320 32 364 398 0.05
Kolangadi
palli
7.66 565 26 404 430 0.04
Gangireddy
palli
734 570 26 444 470 0.00
Basine palli 7.71 460 34 432 466 0.10
** All the values represented in ppm (Parts per million)
73
Table 5: Fluoride contents in serum and urine samples of theselected ten villages people in and around Udayagirimandal (Nellore district, A.P., India)
Name of the
village
Fluoride
content in
Serum in ppm
Fluoride
content in
Urine in ppm
Turkapalli 1.47 2.13
Pakeerpalem 2.1 2.22
Varikunta padu 2.2 4.00
Bijjam palli 1.5 2.12
Masi peta 1.91 1.07
Singa reddy palli 1.05 2.10
Boda banda 2.19 1.23
Kolangadi palli 2.27 2.26
Gangireddy palli 2.13 2.0
Basine palli 2.1 2.12
74
Table 6: Analysis of the blood pressure, random blood sugar andserum creatinine of the normal and fluoride affectedpeople
Blood
Pressure
Random Blood
Sugar
Serum
Creatinine
Control (n=50) 120/80±10 173.58±15.83 1.43±0.35
Fluoride affected
(n=90)
130/90±10 175.59±18.06 2.78±0.24
SEM NS 3.475NS 0.412
Significance P<0.001 P<0.001 P<0.001
NS: Non-significant
75
Table 7: Analysis of the hematological (complete blood picture)parameters of the normal and fluoride affected
people.
WBC RBC Hb% HCT MCV MCH
Control (n=50) 5.20±1.89 4.76±0.49 12.65±1.79 41.92±10.82 92.16±7.59 26.51±1.61
Fluoride affected
(n=90) 6.05±1.84 4.66±0.53 11.91±1.93 38.46±5.11 81.97±4.94 25.15±2.40
SEM 0.403 0.105 0.382 2.308 1.619 0.345
Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
76
Table 8: Analysis of the hematological (complete blood picture)parameters of the normal and fluoride affected people.
MCHC PLT RDW-SD RDW-CV PP W MPV
Control (n=50) 28.86±1.62 151.47±65.27 48.19±4.63 14.81±0.78 13.75±2.81 11.66±1.23
Fluoride affected
(n=90) 30.85±1.47 198.22±76.43 42.16±2.33 13.83±2.67 10.89±2.01 10.15±3.23
SEM 0.345 13.916 0.987 0.168 0.857 0.588
Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
77
Table 9: Analysis of the hematological (complete blood picture)parameters of the normal and fluoride affected people.
P-LCR LYM% MXD% NEUT LYM# MXD# NEUT#
Control
(n=50) 40.95±10.47 38.62±12.36 2.57±1.80 41.50±11.93 2.04±0.79 0.14±0.12 2.25±1.36
Fluoride
affected
(n=90) 31.78±15.81 44.26±11.74 8.58±4.70 37.64±17.46 2.73±0.77 0.51±0.28 2.51±1.70
SEM 2.868 2.635 0.462 5.659 0.169 0.0294 0.378
Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
78
Table 10: Analysis of the lipid profile parameters of the normal andFluoride affected people.
Triglycerides VLDL HDL LDL Cholesterol
Control (n=50) 122.72±36.78 24.53±7.35 41.2±4.45 78.2±25.39 147.4±24.11
Fluoride affected
(n=90) 146.96±66.76 30.1±14.59 39.12±4.71 104.13±26.74 164.09±41.56
SEM 13.352 2.918 0.942 5.348 8.312
Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
79
Table 11: Analysis of the liver function tests of the normal andFluoride affected people.
Control (n=50) Fluoride affected (n=90) SEM SignificanceTotal Bilirubin
(mg/dL) 0.818±0.09 0.829±0.080.0147 P<0.001
Indirect(mg/dL) 0.168±0.03 0.167±0.03
0.00606 P<0.001
Direct(mg/dL) 0.654±0.79 0.664±0.06
0.0121 P<0.001
ALP (U/L) 144.04±30.42 155.14±25.84 4.973 P<0.001SGOT (IU/L) 31.71±3.21 30.18±3.43 0.660 P<0.001SGPT (U/L) 30.20±3.67 27.96±3.31 0.637 P<0.001Total Protein
(g/L) 6.63±0.49 6.87±0.450.0871 P<0.001
SerumAlbumin (g/L) 3.07±0.53 3.69±0.39
0.0745 P<0.001
SerumGlobulin (g/L) 3.49±0.74 3.40±0.86
0.166 P<0.001
A/G Ratio 0.92±0.28 1.14±0.17 0.0327 P<0.001
80
Table 12: Analysis of the electrolytes (sodium and potassium) ofthe normal and fluoride affected people.
Sodium Potassium
Control (n=50) 139.81±4.04 4.04±0.35
Fluoride affected (n=90) 134.37±3.41 4.64±0.45
SEM 0.656 0.0865
Significance P<0.001 P<0.001
81
Table 13: Analysis of the glomerular and tubular markers in the serum of the control and test subjects.
Transferrin IgGα-1-
AntitrypsinBeta-2 MG
g/ml ACEControl(n=50)
131.92±179.87
1742.86±160.51 2.33±17.99
3.03±0.9944.97±8.72
Fluorideaffected(n=90)
123.89±224.93
1364.96±299.73 2.27±16.81 10.60±2.08 37.07±12.68
SEM 43.288 57.684 3.234 0.400 2.441Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
82
Table 14: Analysis of the glomerular and tubular markers in the urine of the control and test subjects.
Transferrin in urine IgG in urine α-1-Antitrypsin Beta-2 MG ACE
Control (n=50) 195.50±29.30 34.54±2.37 36.61±8.38 1.24±0.98 11.46±0.84
Fluoride affected
(n=90) 221.43±49.48 45.41±7.71 39.96±6.54 3.64±0.97 13.77±1.46
SEM 9.352 1.457 1.236 0.183 0.276
Significance P<0.001 P<0.001 P<0.001 P<0.001 P<0.001
83
Table 15: Distribution of the genotype and allele frequencies in theStudy groups for the angiotensin converting enzyme(ACE) I/D polymorphism.
Population
(n)
Genotype frequencies
(percentage)
Allele frequency
DD ID II D allele I allele
Control
(n=60)
10
(16.6%)
33
(55.0%)
17
(28.3%)
0.441 0.559
Fluoride
affected
(n= 90)
16
(17.7%)
48
(53.3%)
26
(28.8%)
0.44 0.56
÷2 based on allele frequency [degrees of freedom (df) = 1], (fluorideaffected Vs Controls) = 0.00025
84
012345678
Turkap
alli
Pakeerp
alem
Variku
nta pa
du
Bijjam pa
lli
Masi p
eta
Singa r
eddy p
alli
Boda
band
a
Kolang
adi p
alli
Gangir
eddy
palli
Basine p
alli
Name of the village
Fluo
ride
conc
entra
tion
in p
pm
Fluoride levels in water
Fig. 5: Comparison of Fluoride content in different villages
85
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Fluoride levels in serum Fluoride levels in urine
Fluoride levels
TurkapalliPakeerpalemVarikunta paduBijjam palliMasi petaSinga reddy palliBoda bandaKolangadi palliGangireddy palliBasine palli
Fig. 6: Analysis of fluoride levels in serum and urine in selectedPopulation from Selected villagers
86
172
172.5
173
173.5
174
174.5
175
175.5
Controls Fluoride affected
population
RBS
in m
g/dl
RBS
Fig. 7: Comparison of Random Blood sugar levels between controls andtest samples
87
Creatinine
0
0.5
1
1.5
2
2.5
3
Controls Fluoride affected
Population
Crea
tinin
e le
vel m
g/dl
Creatinine
Fig. 8: Comparison of serum creatinine levels
88
0102030405060708090
100
WBC RBC HB% HCT MCV MCH
Haematological parameters
Leve
ls ControlsFluoride affected
Fig. 9. Copleate analysis of haematological parametres
89
0
50
100
150
200
250
MCHC PLT RDW-SD
RDW-CV
PPW MPV
Haematological parameters
leve
ls ControlsFluoride affected
Fig. 10. Copleate analysis of haematological parametres
90
05
101520253035404550
P-LCR
LYM%
MXD%NEUT
LYM#
MXD#
NEUT#
Haematological parameters
Leve
ls ControlsFluoride affected
Fig. 11. Copleate analysis of haematological parametres
91
020406080
100120140160180
Triglyc
erides
VLDL
HDLLD
L
Choleste
rol
Lipid profile
Leve
ls in
mg/
dlControlsFluoride affected
Fig. 12. Comparision of lipid profile between controls and fluorideaffected villagers
92
0
20
40
60
80
100
120
140
SBP DBP
Blood pressure
Leve
ls in
mm
of H
gControlsFluoride affected
Fig. 13. Comparision of Blood pressure between controls and fluorideaffected villagers
93
00.10.20.30.40.50.60.70.80.9
Total Bilirubin Direct Bilirubin IndirectBilirubin
Parameters
Bilir
ubin
in m
g/dl
ControlsFluoride affected
Fig. 14. Comparision of bilirubin levels between controls and flourideaffected villagers
94
0
20
40
60
80
100
120
140
160
180
ALP SGOT SGPT
Type of enzyme
Leve
ls in
IU/L
ControlsFluoride affected
Fig. 15. Analysis of liver enzynes between controls and fluoride affectedvillagers
95
0
1
2
3
4
5
6
7
8
Totalprotein
Albumin Globulin A/G
Parameter
Conc
entra
tion
in g
/LControlsFluoride affected
Fig. 16. Comparision of serum protein profile between controls andfluoride affected villagers
96
0
20
40
60
80
100
120
140
160
Sodium Potassium
Type of Electrolytre
Leve
ls in
ku/
LControlsFluoride affected
Fig. 17. Comparision of levels of electrolytes between controls andfluoride affected villagers
97
IgG
11801200122012401260128013001320134013601380
Controls Fluoride affected
IgG
c on
cent
ratio
n in
mg/
dl
IgG
Fig. 18. Comparision of serum IgG levels between controls andfluoride affected villagers
98
0
20
40
60
80
100
120
140
Transferrin antitrypsin BMG ACE
Name of the marker
conc
entra
tion
ControlsFluoride affected
Fig. 19. Comparision of serum marker levels between controls andfluoride affected villagers
99
Comparison of selected markers levels in urine
0
50
100
150
200
250
Transfer
rin IgG ACE
Type of marker
Qua
ntity
of m
arke
r in
mg/
L
ControlFlouride affected
Fig. 20. Comparision of selected urine tubular and glomerular markersbetween controls and fluoride affected villagers
100
Fig.21. Agarose gel electrophoresis stained with ethidium bromide,showing the initial amplification for ACE I/D polymorphism. Lane Lrepresents the 100 bp ladder. The II genotype for I allele wasidentified by the presence of single 490 bp product (Lanes 1, 3, 4and 7). The DD genotype for D allele was identified by the presenceof a single 190 bp product (Lanes 2, 5 and 6). The DD homozygoteswere reconfirmed with insertion specific primer pair to avoidmistyping as ID heterozygotes.
101
Fig. 22. Agarose gel electrophoresis of PCR products, usinginsertion specific primer pair, of individuals labeled as DDhomozygotes following initial amplification. Absence of a productin the lanes 2, 4 and 7 confirms the presence of DD genotype.Heterozygous individuals (ID genotypes) were confirmed by thepresence of a single 275 bp product (Lanes 1, 3, 5, 6 and 8). Lane Lrepresents the 100 bp ladder.