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Biomedicine & Preventive Nutrition 2 (2012) 294–302

Available online at

www.sciencedirect.com

riginal article

eta-analysis of electrolyte imbalance in human fluorosis

onika Bhardwaj ∗, Aggarwal Shashiepartment of Zoology and Environmental Sciences, Punjabi University, Patiala, 147 002 Punjab, India

r t i c l e i n f o

rticle history:eceived 28 April 2012ccepted 12 August 2012

eywords:igh fluorideypocalcemiaodiumotassiumhosphorus

a b s t r a c t

Fluoride is the most electro-negative of all elements. Ingestion of excess fluoride, most commonly indrinking water, can cause fluorosis which affects the teeth and bones. Moderate amount lead to den-tal effects, but long-term ingestion of large amounts can lead to potentially severe skeletal problems.The objective of the present study was to elucidate the effect of high ingestion of fluoride in drinkingwater on electrolyte metabolism in fluorotic patients, selected from seven endemic high fluoride (5.9 to24.5 mg/L) areas of Punjab, India. The levels of calcium, sodium, potassium and phosphorus were deter-mined in serum using kit assay method. Age and sex matched controls consuming water fluoride withinpermissible limit (0.5–1.0 mg/L) were included in the study. The data indicated that the serum levelof calcium declined significantly (F 7,997 = 267.41, P < 0.05 to 0.001) whereas sodium ion concentrationincreased significantly (F 7,997 = 199.09, P < 0.05 to 0.001) in fluorotic patients among all fluoride groups.Potassium and phosphorus level was also increased significantly (P < 0.05 to 0.001) in fluorotic patients

when compared with control group. There was a significant (r = –0.86, P < 0.05) negative relationshipbetween serum fluoride and calcium levels. Correlation analysis indicated highly significant (P < 0.05)positive correlation between serum fluoride and sodium (r = 0.97), potassium (r = 0.90), and phosphorus(r = 0.76) in fluorotic patients.Conclusions: High fluoride ingestion through drinking water could induce hypocalcemia, hypernatremia,hyperkalemia and hyperphosphatemia in patients of fluorosis.

. Introduction

Endemic fluorosis in rural India occurs because of prolongedngestion of water with excess fluoride (> 1 mg/L) resulting inignificant skeletal morbidity. Rural population in addition havether nutritional deficiencies in particular calcium owing to dietaryeficiency. The toxic effects of fluoride on skeletal system are mul-ifactorial reduced intestinal calcium absorption and altered boneemodeling. Calcium absorption through intestine and excretionhrough kidney is tightly regulated. Electrolytes like sodium, potas-ium, and calcium are the important markers of kidney function.hosphorus, an important bone marker is present as inorganichosphate in the body and is complexed with calcium and magne-ium in the bone and teeth. Fluoride ion acts by inhibiting sodiumotassium ATPase that stimulate sodium and calcium exchangend conduces to increase kalemia and also inhibits potassiumransport in human erythrocytes [1]. Fluoride combines with cal-

ium to form calcium ionospheres that easily permeablize the cellembrane. The effect of fluoride depends on extracellular cal-

ium and can be blocked by a combination of calcium channel

∗ Corresponding author. Tel.: +017 53 04 63 34; fax: +017 53 04 63 35.E-mail address: monikabali81@yahoo.com (M. Bhardwaj).

210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved.ttp://dx.doi.org/10.1016/j.bionut.2012.08.009

© 2012 Elsevier Masson SAS. All rights reserved.

blocking agents, suggesting that potentiation of channel activity isdependent on external calcium [2]. Increased levels of phosphorusis seen in chronic nephritis, which progress to renal failure [3]. Thepresent investigation was evaluate the effect of different levels ofhigh fluoride on serum electrolytes and correlation between theseparameters.

2. Materials and methods

2.1. Clinical study

Study group consisted of 705 patients (Males = 393,Females = 312) with clinical defined skeletal fluorosis in the agegroup of 20 to 60 years (mean age of 39.35 ± 11.27) were recruitedfrom seven high fluoride areas (5.9 to 24.5 mg/L) and comparedwith age and sex matched 300 controls. The study protocol wasapproved by the Institutional Human Ethical Committee.

2.2. Biochemical estimations

Blood samples of the subjects were collected by venipunctureinto vacutainers. Blood was centrifuged at 2000 to 3000 rpm for15 minutes to separate out the serum. Serum calcium, sodium,

M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302 295

5

5.3

5.6

5.9

6.2

6.5

6.8

7.1

7.4

7.7

8

8.3

8.6

8.9

9.2

9.5

9.8

Se

rum

ca

lciu

m (

mg

/dl)

0.9 5.9 6.5 7.9 10.5 11 12.2 24.5

Fluoride concentration in drinking water (mg/L)

Fl

pm

2

aop(c

3

3

3

itfpitowPcflt9p

(cdtwil(aafl

0

1

2

3

4

5

6

7

8

9

10

11

12

Control F 1 F 2 F 3 F 4 F 5 F 6 F 7

Study groups

Se

rum

ca

lciu

m (

mg

/dl)

co

nc

en

tra

tio

n 20-30 30-40 40-50 50-60

Fig. 2. Concentration of calcium in serum of control and fluorotic patients of differ-ent age groups.

y = -0.0876x + 8.7032

R2 = 0.5628

r = - 0.75

0

2

4

6

8

10

12

0 5 10 15 20 25 30

Water F (mg/L)

Calc

ium

(m

g/d

l)

7,997 = 199.09, P < 0.0001) variance in the serum level of sodium incontrol and fluorotic patients (Table 5). Bonferroni multiple com-parison test revealed that the serum concentration of sodium ionincreased significantly (t = 10.49 to 27.31, 95% CI = 7.80 to 32.82,

y = - 2.6943x + 9.0617

R2 = 0.7331

r = - 0.86

0

2

4

6

8

10

12

Calc

ium

(m

g/d

l)

ig. 1. Effect of different water fluoride concentration on serum calcium (mg/dL)evel in control and fluorotic patients of endemic fluoride areas.

otassium and phosphorus was analysed by using standard kitethod on semiautomatic analyzer.

.3. Statistical analysis

All continuous data were represented as mean ± standard devi-tion (SD). Statistical tests for significance were performed by usef Anova with subsequent Tukey–Kramer and Bonferroni multi-le comparison tests using Statistical Analysis System softwareversion 9.0, SAS Institute, Cary, NC). A difference at P < 0.05 wasonsidered statistically significant.

. Results

.1. Biochemical analysis

.1.1. CalciumBone and teeth are rich in calcium. Calcium is essential for liv-

ng organisms, particularly in cell physiology, where movement ofhe calcium ion into and out of the cytoplasm functions as a signalor many cellular processes. More than 90% calcium in the body isresent in the bones. Though rest of the calcium, which is present

n plasma is a small amount, it plays a significant role in body func-ions. The mean level of serum calcium in control and patientsf fluorosis decline in all study group (Table 1). One way Anovaith post hoc analysis revealed a highly significant (F 7,997 = 267.41,

< 0.0001, Table 2) difference in the serum concentration of cal-ium in controls and fluorotic patients with the increase of wateruoride levels. Tukey–Kramer multiple comparison test revealedhat the level of calcium declined significantly (q = 30.60 to 42.18,5% CI = –1.63 to –2.93, P < 0.05 to 0.001, Table 1, Fig. 1) in fluoroticatients of all study groups as well as when compared with control.

One way Anova analysis further described highly significantF = 51.03–120.99, P < 0.0001, Table 3, Fig. 2) reduction in serum cal-ium in fluorotic patients of all age groups. Maximum variance ofecline has been noted in 40-50 year age group. Pearson’s correla-ion demonstrated highly significant negative relationship betweenater fluoride and calcium concentration (r = –0.75, P < 0.05, Fig. 3)

n fluorotic patients. High fluoride ingestion through drinking watereads to hypocalcium in fluorotic patients. There was a significant

r = –0.86, P < 0.05) negative relationship between serum fluoridend calcium levels. The regression equation for serum fluoridend calcium was Y = –2.6943x + 9.0617 (Fig. 4). High level of serumuoride in fluorotic patients was also associated with low level of

Fig. 3. Correlation and regression between water fluoride concentration and serumcalcium levels.

serum calcium. Calcium showed inverse relationship with serumfluoride.

3.1.2. SodiumHuman body contains about 150 to 200 gm of sodium. Sodium

is an important electrolyte in the human body. The concentrationof sodium in serum of fluorosis patients was found to be high (7.3%to 21.9%). The increase in mean concentration of sodium ion in flu-orotic patients compared to controls are displayed in Table 4 andFig. 5. One way Anova with post Hoc test showed a significant (F

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Seum fluoride (mg/L)

Fig. 4. Correlation and regression between serum fluoride concentration and serumcalcium levels.

296 M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302

Table 1Levels of calcium (mg/dL) in serum of control and patients affected with fluorosis.

Study group N Water F mg/L Calcium (mg/dl)Mean ± SD

q 95% CI % age decline

Control 300 0.9 9.67 ± 0.27F 1 105 5.9 8.14 ± 0.12a 30.60 –1.63–2.16 –15.82F 2 95 6.5 7.67 ± 0.40ab 32.38 –1.81–2.36 –20.68F 3 105 7.9 7.43 ± 0.21ab 37.22 –2.04–2.57 –23.16F 4 95 10.5 7.76 ± 0.29abd 30.65 –1.70–2.25 –19.75F 5 105 11.0 7.51 ± 0.22abce 40.61 –2.36–2.90 –22.34F 6 100 12.2 7.44 ± 0.18ab 34.82 –1.93–2.47 –23.06F 7 100 24.5 7.05 ± 0.49abcdefg 42.18 –2.39–2.93 –27.09

N: no of control and fluorotic patients in study groups; F 1 – F 7: fluorotic groups; F: Fluoride; q: Tukey–Kramer test value. Means designated with different letters abcdefgare significantly different, P < 0.05–0.001 among study groups.

Table 2One way Anova for serum calcium (mg/dL) concentration in control and fluorotic patients.

Source of variation df Sum of squares Mean square F ratio Significance

Between Groups 7 1123.4 160.48 267.41 P < 0.0001Within Groups 997 595.33 0.60

Total 1004 1718.7

Table 3Analysis of variance for serum calcium concentration in control and fluorotic patients according to age.

Age groups (years) Source of variation df Sum of squares Mean square F ratio Significance

20–30 Between groups 7 197.09 28.156 51.03 P < 0.0001Within groups 227 125.23 0.5517Total 234 322.32

30–40 Between groups 7 175.68 25.098 67.96 P < 0.0001Within groups 251 92.691 0.3693Total 258 268.38

40–50 Between groups 7 363.47 51.924 120.99 P < 0.0001Within groups 260 111.59 0.4292Total 267 475.05

50–60 Between groups 7 386.43 55.205 80.26 P < 0.0001Within groups 235 161.64 0.6878Total 242 548.07

Table 4Serum concentrations of sodium (mMol/L) in control and fluorotic patients exposed to different levels of fluoride in drinking water.

Study group N Water F mg/L Sodium (mMol/L)Mean ± SD

t 95% CI % age increase

Control 300 0.9 143.63 ± 4.58F 1 105 5.9 154.05 ± 5.24a 10.49 7.80–14.43 + 7.25F 2 95 6.5 158.91 ± 8.52ab 13.45 11.34–18.22 + 10.64F 3 105 7.9 168.51 ± 1.68abc 22.81 20.83–27.47 + 17.32F 4 95 10.5 167.51 ± 2.54abc 20.99 19.63–26.52 + 16.63

5 ± 1 abcde

5 ± 11 ± 3

Pg

(tos

TO

F 5 105 11.0 171.2F 6 100 12.2 171.3F 7 100 24.5 175.2

< 0.05 to 0.001, Table 4) in fluorotic patients among all fluorideroups as well as when compared with control group.

One way Anova analysis further described highly significant

F = 30.62–87.79, P < 0.0001, Table 6) increase in sodium concentra-ion in fluorotic patients of different age groups. Maximum variancef increase has been noted in 40- to 50-year age group (Fig. 6). Pear-on’s correlation showed significant (P < 0.05) positive relationship

able 5ne way Anova for sodium ion (mMol/L) concentration in control and fluorotic patients.

Source of Variation df Sum of squares

Between Groups 7 121549

Within Groups 997 86867

Total 1004 208416

.81 26.03 24.33–30.99 + 19.23

.79abcde 26.05 23.54–30.97 + 19.30

.15abcdefg 27.31 26.07–32.82 + 21.99

(r = 0.82) between water fluoride and serum sodium concentrationin fluorotic patients (Fig. 7) and also between serum fluoride andsodium (r = 0.97, P < 0.01) in fluorotic patients (Fig. 8).

3.1.3. PotassiumHuman body contains about 250 gm of potassium. Most of it

(90%) is present in various cells of the body. Remaining is present

Mean square F ratio Significance

17364 199.09 P < 0.000187.21

M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302 297

Table 6Analysis of Variance for sodium level in control and fluorotic patients according to age.

Age groups (years) Source of variation df Sum of squares Mean square F ratio Significance

20–30 Between groups 7 39939 5705.6 30.62 P < 0.0001Within groups 227 42295 186.32Total 234 82235

30–40 Between groups 7 37864 5409.1 70.37 P < 0.0001Within groups 251 19292 76.861Total 258 57156

40–50 Between groups 7 36740 5248.5 87.79 P < 0.0001Within groups 260 15544 59.785Total 267 52284

50–60 Between groups 7 18852 2693.2 38.65 P < 0.0001Within groups 235 16374 69.675Total 242 35226

Table 7Serum concentrations of potassium (mMol/L) in control and fluorosis patients from endemic fluoride areas.

Study group N Water F mg/L Potassium (mMol/L)Mean ± SD

q 95% CI % age increase

Control 300 0.9 4.17 ± 0.42F 1 105 5.9 5.46 ± 0.47a 6.88 0.44–1.91 + 30.94F 2 95 6.5 5.67 ± 0.51a 8.01 0.65–2.15 + 35.97F 3 105 7.9 5.96 ± 1.82a 9.84 0.95–2.41 + 42.93F 4 95 10.5 6.19 ± 1.69ab 10.95 1.17–2.67 + 48.44F 5 105 11.0 6.56 ± 1.41abc 13.42 1.56–3.03 + 57.31F 6 100 12.2 7.82 ± 1.89abcdef 16.75 2.14–3.62 + 87.53F 7 100 24.5 9.67 ± 1.38abcdefg 25.28 3.63–5.12 + 131.89

Fig. 5. Effect of different fluoride concentration on serum level of sodium (mMol/L)in control and fluorotic patients living in high fluoride areas.

0

20

40

60

80

100

120

140

160

180

200

Control F 1 F 2 F 3 F 4 F 5 F 6 F 7

Study groups

Level o

f so

diu

m (

mM

ol/L

) in

seru

m

20-30 30-40 40-50 50-60

Fig. 6. Sodium concentration in serum of control and fluorotic patients of differentage groups.

y = 1.2692x + 151.21

R2 = 0.666

r = 0.82

-20

10

40

70

100

130

160

190

220

0 4.5 9 13.5 18 22.5 27

Water F (mg/L)

So

diu

m (

mM

ol/L

)

Fig. 7. Correlation and regression between water fluoride levels and sodium con-centration.

in extracellular fluids. Potassium is required in the body to storeglycogen in the liver and muscle and for growth of tissues. Table 7presents the toxic effect of fluoride on serum potassium ion con-centration in control and fluorotic patients. Patients of skeletalfluorosis residing in high fluoride drinking water areas, showedsignificant (P < 0.001) increase in their serum potassium ions lev-els as compared to control. Percent elevation of 30.9% to 87.5% inserum potassium concentration were noted at fluoride levels of5.9–12.2 mg/L. Maximum percent elevation of 131.9% was recordedin study group F 7.

One way Anova with post-hoc test showed a significant (F7,997 = 102.43, P < 0.0001, Table 8) difference in the serum levelof potassium. Tukey–Kramer multiple comparison test depictedthat serum concentration of potassium ion increased significantly(q = 6.88 to 25.28, 95% CI = 0.44–5.12, P < 0.05 to 0.001, Table 7) influorotic patients among all fluoride exposed groups as well aswhen compared with control group. The hyperkalemia indicates

that kidneys are unable to filter potassium ions. The enhance-ment in mean concentration of serum potassium in fluoroticpatients when compared to control is shown in Fig. 9. The level

298 M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302

Table 8One way Anova for potassium ion (mMol/L) concentration in serum of control and fluorotic patients.

Source of Variation df Sum of squares Mean square F ratio Significance

Between Groups 7 1782.8 254.68 102.43 P < 0.0001Within Groups 997 2478.9 2.48

Total 1004 4261

Table 9Analysis of variance for serum potassium level in control and fluorotic patients according to age.

Age groups (years) Source of variation df Sum of squares Mean square F ratio Significance

20–30 Between groups 7 271.33 38.762 24.14 P < 0.0001Within groups 227 364.35 1.605Total 234 635.35

30–40 Between groups 7 210.04 30.005 19.45 P < 0.0001Within groups 251 387.17 1.543Total 258 597.17

40–50 Between groups 7 258.17 36.881 26.96 P < 0.0001Within groups 260 355.68 1.368Total 267 613.68

50–60 Between groups 7 230.53 32.933 21.78 P < 0.0001Within groups 235 355.31 1.512Total 242 585.84

y = 40.713x + 145.25

R2 = 0.9437

r = 0.97

0

20

40

60

80

100

120

140

160

180

200

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Serum fluoride (mg/L)

So

diu

m (

mM

ol/L

)

Fe

oaclt

F(

0

1

2

3

4

5

6

7

8

9

Control F 1 F 2 F 3 F 4 F 5 F 6 F 7

Study groups

Seru

m P

ota

ssiu

m (

mM

ol/

L)

20-30 30-40 40-50 50-60

ig. 8. Correlation and regression between serum fluoride content and sodium lev-ls.

f serum potassium in fluorotic patients was highly elevated in

ll age groups and One way Anova test further described signifi-ant (F = 19.45–26.96, P < 0.0001, Table 9, Fig. 10) variance in theirevel. Simple linear correlation and regression analysis revealedhat there was a highly significant (P < 0.05) positive relationship

ig. 9. Effect of different levels of fluoride on serum concentration potassiummMol/L) in control and fluorotic patients of high fluoride areas.

Fig. 10. Potassium ion concentration in serum of control and fluorotic patients ofdifferent age groups.

(r = 0.98, Fig. 11) between water fluoride levels and potassiumions. Fluorotic patients with high serum fluoride levels had highpotassium concentration in serum. Pearson’s correlation test

y = 0.2353x + 4.1025

R2 = 0.9513

r = 0.98

0

2

4

6

8

10

12

0 5 10 15 20 25 30

Water Fluoride (mg/L)

Po

tassiu

m (

mM

ol/L

)

Fig. 11. Correlation and regression between serum fluoride content and potassiumion concentration.

M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302 299

y = 5.8527x + 3.7701

R2 = 0.8107

r = 0.90

0

2

4

6

8

10

12

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

fluoride (mg/L)

Po

tassiu

m (

mM

ol/L

)

m fluoride content and potassium ion concentration.

sp

3

pabwrirp7

7ot1pcsi(rLy

Fp

2.52.83.13.43.7

44.34.64.95.25.55.86.16.46.7

77.37.6

Control F 1 F 2 F 3 F 4 F 5 F 6 F 7

Study groups

Seru

m P

ho

sp

ho

rus(m

g/d

l) le

ve

l 20-30 30-40 40-50 50-60

Fig. 14. Serum level of phosphorus (mg/dL) in control and fluorotic patients of

Serum

Fig. 12. Correlation and regression between seru

howed positive correlation (r = 0.90) between serum fluoride andotassium ions (Fig. 12).

.1.4. PhosphorusHuman body contains 500 to 700 gm of phosphorus. In the body,

hosphorus is present as inorganic phosphate in the bone and teethnd as organic phosphate associated with phospholipids of mem-ranes, nucleic acids and metabolites. Low calcium is associatedith high phosphorus. The mean concentration of serum phospho-

us (mg/dL) in serum of fluorotic patients and controls is presentedn Table 10. The level of phosphorus was found to be high in fluo-otic patients exposed to high fluoride in drinking water. The lowestercent elevation of 24% was noted in fluorotic patients exposed to.9 mgF/L and it raised to highest level of 60.9% at 24.5 mgF/L.

One way Anova with post-hoc test showed a significant (F,997 = 56.56, P < 0.0001, Table 11) difference in the serum levelf phosphorus. Tukey–Kramer multiple comparison test revealedhat the level of phosphorus increased significantly (q = 17.54 to9.95, 95% CI = 0.44–2.28, P < 0.05 – 0.001, Table 10) in fluoroticatients among all study groups as well as when compared withontrol group. The effect of fluoride on the mean concentration oferum phosphorus in fluorotic patients and control, is presentedn Fig. 13. One way Anova test demonstrated highly significant

F = 13.80-36.07, P < 0.0001, Table 12) variance in serum phospho-us level of fluorotic patients and control with increasing age.east variance in phosphorus level has been recorded in 50 to 60ear age group of fluorotic patients (Fig. 14). Pearson’s correlation

ig. 13. Effect of different water fluoride concentration on the serum levels of phos-horus (mg/dl) in control and fluorotic patients.

different age groups.

showed the significant (P < 0.05) and positive relationship (r = 0.78,Fig. 15) between water fluoride ingestion and serum phosphorus inpatients of fluorosis. Linear regression analysis also demonstratedextremely significant positive relationship between serum fluoride

and phosphorus levels (Regression equation: Y = 2.0605x + 4.2984,R2 = 0.57, r = 0.76, Fig. 16).

y = 0.0788x + 4.4549

R2 = 0.6132

r = 0.78

0

1

2

3

4

5

6

7

0 4.5 9 13.5 18 22.5 27

Water F (mg/L)

Ph

osp

ho

rus (

mg

/dl)

Fig. 15. Correlation and regression between water fluoride content and serum phos-phorus level in fluorotic patients.

300 M. Bhardwaj, A. Shashi / Biomedicine & Preventive Nutrition 2 (2012) 294–302

Table 10Serum level of phosphorus (mg/dL) in control and patients affected with fluorosis exposed to different levels of fluoride in drinking water.

Study group N Water F mg/L Phosphorus (mg/dL)Mean ± SD

q 95% CI % age increase

Control 300 0.9 3.81 ± 0.25F 1 105 5.9 5.50 ± 0.20a 17.54 1.29–2.02 + 44.36F 2 95 6.5 5.33 ± 0.15a 15.20 1.04–1.86 + 39.89F 3 105 7.9 4.73 ± 0.32abc 9.06 0.44–1.23 + 24.15F 4 95 10.5 5.51 ± 0.33ad 16.38 1.16–1.98 + 44.62F 5 105 11.0 5.59 ± 0.06ad 18.53 1.32–2.11 + 46.72F 6 100 12.2 5.30 ± 0.43ad 14.19 1.24–1.95 + 39.11F 7 100 24.5 6.13 ± 0.48abcdefg 19.95 1.47–2.28 + 60.89

Table 11One way Anova for serum phosphorus (mg/dL) level in control and fluorotic patients.

Source of variation df Sum of squares Mean square F ratio Significance

Between Groups 7 524.85 74.97 56.56 P < 0.0001Within Groups 997 1316.35 1.32

Total 1004 1841.2

Table 12Analysis of variance for serum phosphorus level in control and fluorotic patients according to age.

Age groups (years) Source of variation df Sum of squares Mean square F ratio Significance

20–30 Between groups 7 209.82 29.974 24.19 P < 0.0001Within groups 227 281.26 1.239Total 234 491.08

30–40 Between groups 7 273.31 39.044 36.07 P < 0.0001Within groups 251 271.68 1.082Total 258 544.99

40–50 Between groups 7 281.42 40.202 30.44 P < 0.0001Within groups 260 343.35 2.321Total 267 624.77

139.3338.9478.3

4

pohawi(

Fs

50–60 Between groups 7

Within groups 235

Total 242

. Discussion

Calcium has always been considered the basic element in vitalrocesses. The significance of the phosphorus-calcium balance andf fluorine in the process of mineralization of bones and teethas also been long known. Low calcium levels is usually associ-ted with high phosphorus. In cells calcium combine with fluoride,

hich results in the formation of calcium fluoride. The element

s deposited in various forms in cells and tissues. Some enzymePeptidase, alpha-amylases, phosphateses), are activated by

y = 2.0605x + 4.2984

R2 = 0.5767

r = 0.76

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Serum fluoride (mg/L)

Ph

osp

ho

rus (

mg

/dl)

ig. 16. Correlation and regression between serum fluoride concentration anderum phosphorus level in fluorotic patients.

9 19.913 13.80 P < 0.00013 1.4422

calcium ions and are inhibited by added fluoride. One of the pos-sible mechanisms of enzyme inhibition in these reactions may becalcium binding to fluoride in catalytic center [4]. As fluoride is aelectronegative element, it tightly binds with many calcium cationsand causes hypocalcaemia [5]. It is well known that poor nutritionand low calcium intake enhance the deleterious effect of fluoride[6]. The resultant hypocalcemia is responsible for various manifes-tation of fluorosis like delayed eruption of teeth, dental, skeletal,and clinical fluorosis as well as premature aging etc. [7].

The present study revealed hypocalcemia in patients of fluo-rosis exposed to different levels of fluoride. The decreased levelsof serum calcium in the fluorotic individuals could be attributed toectopic calcification in soft tissues. It could also be due to a decreasein the intestinal absorption of fluoride since fluoride is known toproduce insoluble complexes with calcium [8]. Our findings are inconsonance with the studies of Gessner et al. [9], Chakma et al. [10],Gupta et al. [11], Ahmed et al. [12], Khandare et al. [13], Harinarayanet al. [3] and Gupta et al. [14], all have reported decreased levels ofserum calcium in the patients of fluorosis.

Li et al. [15] found a significant decrease in mean plasma cal-cium concentration with increased fluoride exposure in populationof apparently healthy adults with inadequate nutrition. With a highfluoride intake, insoluble calcium fluoride formed in the intestineand excreted in faeces, increasing the likelihood of a low blood cal-cium if there is an insufficient dietary intake. In turn, hypocalcaemia

may lead to parathyroid stimulation with secondary hyperparathy-roidism, bone matrix resorption, osteoporosis and osteomalacia[16]. It was well known that ionic calcium was one of the importantions for the initiation and maintenance of the activity of the vital

& Pre

owhlc

aspeecacoitcwtiarHwadm

epccnsiaaacamor6iuSswG

ltsftkcecplacr

M. Bhardwaj, A. Shashi / Biomedicine

rgans and musculo-skeletal system. Lowering of ionized calciumas one of the important stimulus for the release of PTH [17]. Theypocalcemia caused by high fluoride ingestion in drinking water

eads to changes in internal milleu of the body to maintain thealcium levels and can cause secondary hyperparathyroidism [11].

Harinarayan et al. [3] reported hypocalcemia raised serumlkaline phosphatase with normal serum phosphorus in patientsuffering from fluorotoxic metabolic bone disease. There was aositive correlation between serum creatinine and phosphorusxcretion index and a negative correlation between decliningndogenous creatinine clearance and increasing renal loss of cal-ium and phosphorus as indicated by calcium to creatinine rationd phosphorus excretion index (PEI). Thus, low levels of calciumould also be the another factor for increase in aggravating problemf genu valgum [10,16] caused increased parathormone levels andncreased osteoblastic activity. The pathogenesis of bone deformi-ies in fluorosis and nutritional rickets caused by calcium deficiencyan be explained as, in fluorosis, the osteoblastic activity increases,hich are indicated by elevated alkaline phosphatase activity,

hereby increasing the calcium demand (calcium deficiency). Thisncreased osteoblastic activity led to excessive formation of osteoidt the growing ends of the bone during calcium deficiency state,esulting in the widening of the epiphysiol ends of metaphysis [13].igher BMD of lumber spine and femur neck in fluorotic subjectsas due to prolonged intake of high fluoride from drinking water

nd poor calcium nutrition [18]. The hypocalcaemia was observedue to decrease calcium absorption from gut, and increased accu-ulation in tissue [19].Differential distribution of sodium and potassium cations is

ssential for normal membrane function and integrity. Serumotassium is an indicator of cell damage. Increased levels suggestsell deterioration. It has been reported that fluoride is known toause potassium efflux [20]. The present study observed hyper-atremia, and hyperkalemia in fluorotic patients. The rise in theodium and potassium ions could be attributed to the alterationsn electrolytes balance in inter or/and intracellular fluids as wells sodium and potassium levels consumed in drinking water. As

result, this might influence the movement of the water insides well as outside the cells. The overall distribution of these twoations is vital in many membrane system where energy orientedctive transport in functional. Similar finding have been docu-ented earlier by authors in their investigations. Michael et al. [8]

bserved the marked alteration in serum electrolyte levels of fluo-otic group. Sodium and potassium levels increased significantly at.53 ppm water fluoride compared to controls. Barot [21], observed

ncreased levels of sodium and potassium ions in fluorotic pop-lation at 2.81 ppm fluoride in water of Ahmedabad city. While,hivashankara et al. [22] reported lowered levels of serum potas-ium and normal sodium ion concentration in children affectedith skeletal fluorosis at 13.4 ppm fluoride in drinking water ofulbarga district, Karnataka, India.

In experimental animals, Kessabi et al. [23] demonstrated highevels of potassium ions and decline in sodium ion concentra-ion. The relatively hyperkalemia observed in the Darmous areaheep might be due to the electrolyte disturbances which resultsrom cachexia, secondary to fluorosis. Moreover, acute fluorideoxicity induced hyperkalemia [24]. Similar finding on hyper-alemia in experimental fluorosis were also observed in sheep,attle [25], and goat [26]. A rise in the plasma potassium lev-ls could be related to renal and adrenal dysfunction [27,28]. Theellular breakdown and protein catabolism can also increase theotassium levels in the blood and diarrhea could further increase

oss of potassium through gastrointestinal tract. Mitsui et al. [29]nd Shanthakumari and Subramanian [19] showed increased con-entration of serum sodium and potassium ions in experimentalats.

ventive Nutrition 2 (2012) 294–302 301

Adachi et al. [30] determined the effect of cadmium fluorideon serum electrolytes and acid-base balance in rats and observedthat serum potassium level increased in a dose dependent mannerand hyperkalemia was further exacerbated by the complication ofacute renal failure. Increased potassium in the NaF treated groupresulted mainly from exposure to the ionized fluoride containedin NaF. Kant et al. [26] documented the decrease in the plasmasodium levels of NaF treated goats. In acute fluoride intoxication,fluoride, the most electronegative element, is well-known to lowerthe levels of cations such as calcium and magnesium, because ofthe tight binding of the fluoride with these cations. Moreover, fluo-ride also inhibits Na+K+ ATPase on the cell surface, thus increasingintracellular Na+, triggering the Na+, Ca2+ exchange and increas-ing the intracellular Ca2+. The subsequent rise intracellular Ca2+

opens Ca2+ – dependent potassium channels, and an explosivepotassium efflux ensues, especially from muscle cells, with a poten-tially lethal effect on the heart [20,31]. Nichloy et al. [1] suggestedthat in chronic administration, fluoride may partially inhibit Na+K+

ATPase. On the other hand, Das and Susheela [32] had shownthat plasma cortisol levels were markedly decreased in fluorosispatients. Both corticosteroids are concerned, authors clearly sug-gested an adrenal hypofunction in chronic fluoride toxicity: so inthe light of this work, it is possible to suspect decrease of aldos-terone, which can explain tendency to hyperkalemia in patientswith high plasma fluoride levels. Furthermore, one other studyshows that sodium fluoride inhibits K+ transport in human erythro-cytes [33]. In vitro studies have shown that potassium efflux fromerythrocytes results when erythrocytes make contact with fluoride[34].

Hyperphosphatemia was observed in human fluorosis revealedthe direct effect of fluoride on the phosphorus level. Serum phos-phorus level is might be due to the high intake of fluoride,as it may cause imbalance in the serum calcium and phospho-rus levels due to fluoride induced Ca chelation. Consequently,phosphorus level increased but calcium level decreased. Hamidand Dorra [35] observed that raised levels of serum phospho-rus at high fluoride intake indicate nephritic dysfunction. Gessneret al. [9] found an increase in levels of serum phosphorus, whenpatients were exposed to high levels of fluoride poisoning indrinking water in Alaska. Increase in serum phosphorus was alsoobserved by Khandare et al. [13] in their work on severe bonedeformities. High phosphorus levels exert a direct effect on thekidney by increasing parathormone production by parathyroid.The severe osteomalacia demonstrated earlier may be due tomineralization defects caused by nephrogenic hypocalcemia andhypophosphatemic defects due to tubular damage as documentedin several reports of fluorotoxicity related renal dysfunction inhumans [3]. Alma et al. [36] also observed an increase in serumphosphorus in fluorotic patients which may due to parathy-roid stimulation. Ahmad et al. [12] reported the lower levelof serum inorganic phosphorus levels in children affected withskeletal fluorosis at 29 ppm water fluoride as compared to nor-mal control group. In another human study by Khandare et al.[18], observed significantly lower level of serum phosphorus influorotic group at 4.5 ppm fluoride in water, in comparison to non-fluorotic group. Similar results of increased serum phosphorus levelwere also observed in cattle [37], rat [19], buffalo [38], and goat[26].

5. Conclusions

There is an association between fluoride toxicity and electrolytedisorders demonstrated by increased levels of sodium, potassiumand phosphorus as well as decreased levels of serum calcium influorotic patients.

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02 M. Bhardwaj, A. Shashi / Biomedicine

isclosure of interest

The authors declare that they have no conflict of interest con-erning this article.

cknowledgement

The financial assistance from Rajiv Gandhi National Fellowshiprogramme, University Grants Commission, Government of India,ew Delhi, is gratefully acknowledged.

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