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.