10.5005/jp-journals-10021-1309
Anuj Satija et al
ORIGINAL RESEARCH
Evaluation of Salivary and Serum Concentration of Nickel
and Chromium Ions in Orthodontic Patients and Their
Possible Influence on Hepatic Enzymes: An in vivo Study
1
Anuj Satija, 2Maninder Singh Sidhu, 3Seema Grover, 4Vikas Malik, 5Puneet Yadav, 6Rohan Diwakar
ABSTRACT
INTRODUCTION
Objectives: The aims of present study were to evaluate nickel
and chromium ions concentrations in salivary and serum
samples from patients treated with fixed orthodontic appliances
and their possible influences on hepatic enzymes.
During the last decade, there has been an increased interest
among orthodontic professionals regarding side effects
associated with use of biomaterials, especially metallic
materials, such as gold, stainless steel, cobalt-chromium,
nickel-titanium and beta-titanium.1 Nickel-titanium alloys
were introduced for use as orthodontic wires in 1970s2
which contain 47 to 50% Ni and thus are the richest source
of nickel among all alloys.3
Oral environment is particularly ideal for the biodegradation of metals because of its ionic, thermal, microbiologic and enzymatic properties.4 In oral environment,
biodegradation of metals usually occurs by electrothermal
breakdown.5 The association of different metals in oral
environment, where saliva is the connecting medium may
produce electrogalvanic currents that produce discharge of
ions and metallic components. Corrosion of alloys in the
oral environment may decrease the strength of metallic
appliances.6 Discoloration on the underlying tooth surface
during orthodontic treatment has also been regarded as one of
the consequences of crevice corrosion of the bracket bases.7
Nickel and chromium have dermatological, toxological and
possibly mutagenic effects.3 It is estimated that 4.5 to 28.5%
of population have hypersensitivity to nickel with higher
prevalence in females.8 Chromium allergy is estimated at
10% in male subjects and 3% in female subjects.9
Nickel hypersensitivity symptoms include rash, allergy,
lung disorder, ear infection and tinnitus. Nickel is most
common cause of metal induced allergic contact dermatitis
in man and second in frequency is chromium.9 In addition
to the allergic issue, carcinogenic, mutagenic and cytotoxic
effects have been assigned to nickel and to a lesser extent
chromium. The most significant method for measurement of
nickel release before and after onset of orthodontic treatment
is salivary analysis, since it is the first diluents of the human
body and allows long period of analyses. Also most of the
redox reactions occur in liver and nickel is a redox active
metal and is able to impair cellular defence mechanisms
against peroxidation reactions.
This is the first in vivo study relating salivary and serum
levels of nickel and chromium to hepatic enzymes. It has
been reported in literature increased serum level of nickel
Materials and methods: Saliva and blood samples were
collected from 36 patients ranging in age from 12 to 24 years.
Three samples of saliva and two samples of blood were
obtained. First saliva and blood samples were collected
before inserting fixed appliances. Second salivary samples
were collected at 1 week, third salivary sample and second
blood sample after 4 weeks of appliance insertion. Serum
was prepared by centrifuging blood samples at 3000 rpm for
10 minutes. Spectrophotometric determinations were carried
out using electrothermal atomic absorption spectrophotometry.
Results and conclusion: In serum, significant increase in Ni
and Cr ion concentration occurred in samples collected after
4 weeks. In saliva samples, nickel and chromium reached their
highest lavels in first week. Mean liver function enzymes SGOT
and SGPT were also significantly increased in 4 weeks. Fixed
orthodontic appliances release measurable amount of nickel
and chromium when placed in mouth, but this increase does not
reach toxic levels for nickel and chromium in saliva and serum
to cause harmful effects in human beings.
Keywords: Nickel, Chromium, Fixed orthodontics, Hepatic
enzymes.
How to cite this article: Satija A, Sidhu MS, Grover S, Malik V,
Yadav P, Diwakar R. Evaluation of Salivary and Serum
Concentration of Nickel and Chromium Ions in Orthodontic
Patients and Their Possible Influence on Hepatic Enzymes:
An in vivo Study. J Ind Orthod Soc 2014;48(4):518-524.
Source of support: Nil
Conflict of interest: None
Received on: 11/7/14
Accepted after revision: 6/8/14
1,5,6
Senior Lecturer, 2Professor and Head, 3Professor, 4Reader
1-6
Department of Orthodontics, SGT Dental College and
Hospital, Gurgaon, Haryana, India
Corresponding Author: Maninder Singh Sidhu, Professor
and Head, Department of Orthodontics, SGT Dental College
and Hospital, Gurgaon, Haryana, India, Phone: 09811312815
e-mail: [email protected]
518
JIOS
Evaluation of Salivary and Serum Concentration of Nickel and Chromium Ions in Orthodontic Patients
have been found to affect lungs, kidney, liver and heart.10
Concentrations of serum nickel have been found to decrease
in hepatic cirrhosis as a consequence of hypoalbuminemia.11
Hence, present study was conducted to measure nickel and
chromium ion concentrations in serum and saliva before and
after insertion of orthodontic appliance and also to evaluate
changes in salivary nickel and chromium ions concentration
during orthodontic treatment and their possible influences the
hepatic enzyme levels. The null hypothesis was that changes
in salivary and serum nickel and chromium will not affect
hepatic enzymes levels.
MATERIALS AND METHODS
This study was conducted at the Department of Orthodontics,
SGT Dental College and Hospital, Gurgaon (India). The sample
size consisted of 36 subjects (21 females and 15 males), who
were to commence their fixed orthodontic treatment and had no
history of having received any prior orthodontic appliance. The
mean age of the sample was 17.5 years (14-24 years).
An informed consent was obtained from all patients who
participated in the study, and ethical clearance was obtained.
The main criteria for subject inclusion were not that subjects
should not be suffering from any systemic disease and
should not have habits of smoking, red wine intake, excess
of tea, coffee or betelnut, tobacco chewing and no amalgam
restorations in oral cavity.
Preadjusted edgewise appliance (ORMCO 0.022" MBT
prescription) was used in all cases. All patients received
0.016" NiTi wire as an initial wire for alignment and
leveling, which was tied into the bracket slot with stainless
steel ligatures. None of the patients had palatal or lingual
appliances welded to the bands or extraoral orthodontic
appliances.
Three samples of saliva were collected from each
orthodontic patient. First sample before insertion of the fixed
appliance which served as a baseline/reference level for
salivary nickel and chromium content, second after 1 week,
and third after 4 weeks of insertion of appliance.
For the collection of saliva, patients initially rinsed
the mouth thoroughly with a mouthful of deionized and
distilled water. The patients, after an initial swallow were
instructed to collect approximately 10 ml of saliva with
mouth closed and released into the plastic test tube. In this
way, approximately 10 ml of saliva was collected into an
acid-washed (concentrated HNO3) plastic test tube. After
collection, the samples were stored in ice box at –20°C in
dry ice until they were processed.
Two samples of blood were collected from each
orthodontic patient that is before insertion of fixed appliance
which served as a baseline/reference level for serum nickel
and chromium content and 4 weeks after insertion of the
appliance. Blood was obtained from antecubital fossa of
arm and centrifuged at 3000 rpm for 10 minutes in order to
prepare serum.
Preparation and Analysis of the Samples of
Saliva and Blood
For processing, 0.5 ml of saliva/serum was transferred to
smaller plastic test tubes (Cryo Tubes, Polylabs, New Delhi)
using a micro-pipette (Biochemical Ltd, Takson, India)
which were pretested for not releasing nickel. For digestion
of organic matter in saliva/serum, 0.15 ml of concentrated
HNO3 was added to each sample. The tubes were then closed
and kept at 80oC for 8 hours in hot air oven. The saliva
samples were centrifuged at 3000 rpm for 2 minutes to settle
particulate matter. The serum samples were centrifuged at
3000 rpm for 10 minutes to settle particulate matter (Fig. 1).
The chemical analyses for nickel and chromium were
done by electrothermal atomic absorption spectrometry
(model AAS5 EA, Analytik Jena Ltd, Germany), at the Vimta
Labs Ltd, Life Sciences Facility, Hyderabad, India (Fig. 2).
Atomic absorption spectrometry (AAS) is an analytical
technique that measures the concentrations of elements. The
technique makes use of wavelengths of light specifically
absorbed by an element. They correspond to energies needed
to promote electrons from one energy level to higher energy
level. The analytic lines used were 357.9 nm for chromium
and 232.0 nm for nickel.
Calibration Curve for Nickel and Chromium (Fig. 3)
A calibration curve was used to determine the unknown
concentration of an element (Ni, Cr) in a solution. A sample
of known concentration of 20, 40, 60, 80, 100 ppm (obtained
The Journal of Indian Orthodontic Society, October-December 2014;48(4):518-524
Fig. 1: Centrifuge machine
519
Anuj Satija et al
Fig. 2: Electrothermal atomic absorption spectrometry (model
AAS5 EA, Analytik Jena Ltd, Germany), at the Vimta Labs Ltd, Life
Sciences Facility, Hyderabad
Fig. 3: The working of atomic absorption spectrometer
Fig. 4: Xenon lamp as a continuous radiation source
Graph 1: A normal calibration graph or calibration curve of
nickel and chromium
by diluting 1000 ppm standard solution from MERCK India)
were subjected to and absorbance (Abs) values were plotted
vs concentration levels. The calibration curve showed the
concentration against the amount of radiation absorbed. If a
linear calibration curve was obtained, the slope of calibration
could be obtained by using following equation:
A = mc (Beer-Lambert law)
Absorbance = slope × concentration
the solvent, 6 seconds at 950oC to drive off any volatile
organic material and change the sample to ash, and with a
fast heating rate to 2100oC for 5 seconds to vaporize and
atomize the elements (Graph 1). Finally, tube was heated to
higher temperature of 2300oC to make it clean and keep it
ready for next sample.
Then a beam of electromagnetic radiation from a hollow
cathode lamp (specific for Ni—232.0 nm and Cr—357.9 nm)
(Fig. 4) was passed through the vaporized sample. Some
of the radiation was absorbed by the metal atoms in the
sample. The standard amount absorbed by metal atoms
was compared with the calibration curve and this enables
calculation of metal (Ni and Cr) concentration in unknown
sample by using Beer-Lambert law:
Methodology and Determination of
Concentration of Ni and Cr in
Saliva and Serum Samples
Twenty microliter of sample was injected directly into
graphite tube (Analytik Jena Ltd, Germany) from an
automated micropipette (Biochemical Ltd, India). The
tube was heated electrically by passing a current of 5.0
milliamperes (mAmp) through it in a pre-programed series
of steps which included 15 seconds at 120°C to evaporate
520
Absorbance (recorded from AAS)
Concentration =
(mg/l)
Slope (calculated from calibration curve)
JIOS
Evaluation of Salivary and Serum Concentration of Nickel and Chromium Ions in Orthodontic Patients
Table 1: Salivary and serum Ni and Cr concentrations (ppb) in individual subjects at different time periods
Salivary Ni
Salivary Cr
Serum Ni
Serum Cr
Pre
1 week
4 weeks
Pre
1 week
4 weeks
Pre
4 weeks
Pre
4 weeks
Mean
5.432
10.372
6.685
0.596
1.509
0.881
8.265
9.948
6.477
9.975
Std. deviation
0.916
0.940
1.053
0.049
0.095
0.043
0.581
0.433
0.274
0.441
Table 2: Paired t-test for salivary nickel and chromium (ppb) concentration
N = 36
Salivary Ni
Pair 1
Pair 2
Pair 3
Salivary Cr
Pair 1
Pair 2
Pair 3
Mean
Std. deviation
t-value
p-value
Pre
5.4323
0.9164
75.977
<0.001*
1 week
10.3727
0.9401
—
—
Pre
5.4323
0.9164
8.903
<0.001*
4 weeks
6.6854
1.0535
—
—
1 week
10.3727
0.9401
27.998
<0.001*
4 weeks
6.6854
1.0535
—
—
Pre
0.596
0.049
50.395
<0.001*
1 week
1.509
0.095
—
—
Pre
0.596
0.049
30.845
<0.001*
4 weeks
0.881
0.043
—
—
1 week
1.509
0.095
44.134
<0.001*
4 weeks
0.881
0.043
—
—
*p < 0.001: Highly significant
Table 3: Paired t-test for serum nickel and chromium (ppb) concentrations
N = 36
Serum nickel
Serum chromium
Mean
Std. deviation
t-value
p-value
Pre
8.265
0.5817
26.703
<0.001*
4 weeks
9.9482
0.4337
—
—
Pre
6.4777
0.2744
57.59
<0.001*
4 weeks
9.9756
0.4417
—
—
*p < 0.001: Highly significant
Table 4: Liver function enzyme levels (U/L) in individual subjects after 4 weeks
SGOT
pre(U/L)
SGOT
4 weeks (U/L)
SGPT
pre (U/L)
SGPT
4 weeks (U/L)
Alkaline
phosphatase
pre (U/L)
Alkaline
phosphatase
4 weeks (U/L)
Mean
30.416
37.944
35.166
43.055
237.972
256.694
Std. deviation
4.964
3.725
3.730
3.097
10.266
10.706
RESULTS
In this study, 36 subjects, mean age of 17.5 years, who were
to commence their fixed orthodontic treatment and had no
history of having received any prior orthodontic appliance,
fixed or removable were selected. The sample distribution
was divided into two groups.
Table 1 shows that mean salivary nickel and chromium
concentrations were increased in all patients after 1 week
compared to baseline and 4 weeks levels. Hence, leaching of
nickel and chromium ions from orthodontic wire, brackets
and bands were maximum at 1 week when compared to
baseline and 4 weeks levels. Also serum levels of nickel and
chromium concentrations were increased in 4 weeks interval.
Table 2 shows paired t-test for salivary nickel and
chromium concentrations. Here, p-value in all three
combination was <0.001, hence there was highly significant
increase in observation taken at pretreatment and at 1 week,
also deviation from pretreatment to 4 week was highly
significant but this increase was not as high as at 1 week.
The normal range for serum nickel have been reported
to be between 6.587 and 10.912 ppb. The normal range
for serum chromium are reported to be between 6.082 and
10.990 ppb.
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Anuj Satija et al
Table 5: Paired t-test for SGOT, SGPT and alkaline phosphatase concentration
N = 36
SGOT
SGPT
Alkaline
phosphatase
Mean
Std. deviation
t-value
p-value
Pre
30.4167
4.9649
15.351
<0.001*
4 weeks
37.9444
3.7259
—
—
Pre
35.1667
3.7302
14.606
<0.001*
4 weeks
43.0556
3.0979
—
—
Pre
237.9722
10.2664
10.865
<0.001*
4 weeks
256.6944
10.706
—
—
*p < 0.001: Highly significant
Table 3 shows paired t-test for serum nickel and
chromium concentrations. The mean value for serum nickel
ions for baseline level was 8.265 ppb and for serum nickel
4 weeks levels was 9.948 ppb. The mean value for serum
chromium ions for baseline level was 6.477 ppb and at
4 weeks levels was 9.975 ppb. The p-value for baseline and
4 weeks was <0.001 which shows statistical significance
difference.
The normal range of SGOT concentrations varies from
18 to 45 U/L and normal range of SGPT concentrations
varies from 28 to 49 U/L.
Table 4 shows that SGOT and SGPT levels were
increased in all patients at 4 weeks interval compared to
baseline group, but they remain within normal range. The
p-value for both at baseline and 4 weeks was <0.001 which
was statistical significant.
The normal range of alkaline phosphatase varies from
208 to 278 U/L. Alkaline phosphatase levels at 4 weeks
were increased in all patients compared to baseline group,
but they remained within normal range.
The p-value at baseline and 4 weeks was <0.001 which
was statistically significant.
Table 5 shows paired t-test for SGOT, SGPT and alkaline
phosphatase concentrations. Here p-value was <0.001, hence
there was highly significant increase in observation taken at
pretreatment to 4 weeks interval.
DISCUSSION
A wide variety of appliances and auxiliaries used in
orthodontics contain nickel and chromium containing alloys,
and thus they have become an integral part of almost every
routine orthodontic intervention.3 The potential health effects
from exposure to these compounds have been scrutinized
for more than 100 years, and sensitizing patients to these
metals have been a concern.12
Nickel has threshold concentration of approximately 30
ppm which may be sufficient to elicit a cytotoxic response.13
Nickel has been found to induce DNA alterations mainly
through base damage and DNA strand scission (single
strand breaks), which are site specific. The mechanism of
522
action of mutations caused by nickel is due to inhibition of
several enzymes known to restore DNA breaks promoting
microsatellite mutations and increasing total genomic
methylation, thereby contributing to genetic instability.14
Nickel toxicity has been reported in literature very well
to the extent of death in few cases and postmortem report
showed nickel level as µg/g wet weight 7.5 in blood, 47 in
urine, 27 in liver, 60 in bile respectively.15
In present study, saliva samples were obtained from
orthodontic patients in order to analyze the release in the
dynamic oral cavity environment. The main advantage of
current in vivo study as compared to the in vitro studies was
that the concentrations of salivary nickel and chromium were
recorded in the natural oral environment of the patient, where
the actual side effects of increased metal concentrations were
going to take place.
The present study when compared with study by Gjerdet
et al16, who conducted a study on nickel and iron in saliva
of patients with fixed orthodontic appliances found no
significant differences in absolute masses of nickel or iron
in samples taken in saliva before, and 3 weeks after insertion
of fixed appliances. But for samples taken immediately
after 1 week, there was a significant increase in both
concentrations and masses of nickel and iron. In the present
study, there was a significant difference found between
the pretreatment samples and the samples taken 1 and
4 weeks.
Kerosuo et al17 reported nickel release of 16.7 µg from a
simulated fixed appliance and 7.5 µg from headgear during
7 days under static conditions. Grimsdottir et al18 conducted
an in vitro study and analyzed the release of nickel from
different types of metal appliances immersed in physiologic
saline and found that amount of metal release was highest
from facebows followed by molar bands, brackets and
archwires (NiTi).
Petoumenou et al 19 concluded that samples taken
immediately after placement of bands, brackets and NiTi
archwires showed slight but significant increases in nickel
concentration of 78 and 56 μg per liter respectively,
compared with pretreatment value of 34 μg per liter. Nickel
JIOS
Evaluation of Salivary and Serum Concentration of Nickel and Chromium Ions in Orthodontic Patients
leaching occurred after placement of bands, brackets and
NiTi archwires, associated with an increase of nickel ion
concentration in patient’s saliva and this effect decreased
within 10 weeks.
The range of serum nickel concentrations increased
mainly from 8.265 to 9.948 ppb 4 weeks after insertion of
fixed appliances (Table 3). There was a significant difference
(p < 0.001) found between the no appliance samples and the
samples taken 4 weeks after bonding and banding tested by
the students t-test.
The range of serum chromium concentration increased
mainly from 6.477 to 9.975 ppb 4 weeks after insertion of
fixed appliances (Table 3). There was a significant difference
(p < 0.001) found between the pretreatment samples and the
samples taken 4 weeks after bonding and banding tested by
the students t-test.
Nickel can be taken into the body by eating food,
drinking water, or breathing air. Average dietary intake
of nickel in adult persons is 165 µg/day but may reach
900 µg/day in diets rich in cocoa, oatmeal, nuts and soya
products.20,21 Only 1 to 5% of ingested nickel is absorbed
in the body, remainder is excreted in the feces.
About 5% of the amount ingested is absorbed into
bloodstream through intestines, while 20 to 35% of inhaled
nicked is absorbed through lungs. Total 68% of nickel
reaching in blood is rapidly metabolized in liver and rest
is excreted in urine, while 2% remains in kidneys with
very short biological half-life of 0.2 days (about 5 hours).22
The remaining 30% is evenly distributed to all remaining
tissues of body, including the kidneys, liver and clears with
a biological half-life of more than 3 years (1,200 days).
Measurements of nickel concentrations in plasma from
retired nickel refinery workers demonstrated that nickel
was retained for several years after termination of nickel
exposures.23
Literature supports direct correlation of nickel concentrations in plasma and urine. 24-26 Also concentrations
of serum nickel are decreased in hepatic cirrhosis, as a
consequence of marked hypoalbuminia. Hepatic toxicity,
manifested by microvesicular steatosis, transient diminution
of hepatic glutathione concentration, increased activity of
serum alkaline phosphatase, develops in nickel chloride
treated rats.
Also it has been reported in literature that release of nickel
in the first 6 months was higher than at rest of time periods.
This could be attributed to maximum appliance degradation
in the initial period of treatment. At the same time, nickel is
more cathodic in nature which may be the cause of initial
release of nickel and chromium. This is in correlation with
the present study findings. As the deformation deactivates,
though the oral environment remains same, the return of the
material more towards normal stage causes gradual decrease
in the release of nickel.27
Urine and serum are the body fluids with widest utility
as specimens for nickel analysis in biological monitoring
programs. Hence, serum was used in the present study to find
out possible systemic effects on liver by observing changes
in SGOT, SGPT and alkaline phosphatase levels.
Hence, we aimed to find out correlation between salivary
and serum levels and further serum sample was used to find
out changes in SGOT, SGPT and alkaline phosphatase levels
to rule out any hepatic changes.
The results of the present study also showed that there
was a statistical difference in SGOT, SGPT and alkaline
phosphatase concentrations before and 4 weeks after
insertion of different fixed orthodontic appliances, but they
remain within normal level.
Natarajan28 evaluated genotoxic effects of fixed appliances on oral mucosal cells and the relationship to nickel
and chromium concentrations which was an in vivo study.
The author concluded that nickel and chromium alloys
of orthodontic appliances emit metal ions in sufficient
quantities to induce localized genotoxic effects, but these
changes revert on removal of appliances.
Hafez29 described cytotoxicity, genotoxicity, and metal
release in patients with fixed orthodontic appliances, which
was a longitudinal in vivo study. The author concluded that
fixed orthodontic appliances decreased cellular viability,
induced DNA damage, and increased the nickel and
chromium contents of the buccal mucosa cells. Compared to
control group, these changes were not evident at 6 months,
possibly indicating tolerance for or repair of the cells and
the DNA.
There are very few studies in the literature showing
systemic influences of Ni and Ti in orthodontic patients.
Hence, future research should be carried for a longer period
of time to study the effects of corrosion processes and
mechanical phenomena, such as wear and fatigue on release
of nickel and chromium in oral cavity. A larger sample size
should also be used to eliminate the variability of results.
CONCLUSION
Following conclusions were drawn from the results of
present study:
• The results indicate that orthodontic appliances corrode
in oral environment and release nickel and chromium.
Although, there is initial rise in salivary and serum nickel
and chromium levels in 1 week but significantly tapered
to permissible blood level in 4 weeks. Hence, this should
The Journal of Indian Orthodontic Society, October-December 2014;48(4):518-524
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Anuj Satija et al
be taken care of in patients having allergy to nickel and
chromium.
Also, there was significant rise in hepatic enzymes level
from baseline to 4 weeks but it was within normal range.
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