Analysis of Metals in Saliva by Atomic Absorption
Spectroscopy. I. Calcium
B. S. GOW*
Institute of Dental Research, United Dental Hospital, Sydney, Australia
The dependence of the tooth for the main-
tenance of its health on the dissolved cal-
cium in the saliva is an established fact. As
both dental caries and the formation of den-
tal calculus may involve the calcium of the
tooth and saliva to a greater extent than
other metals, a full knowledge of the chemis-
try of oral calcium and of a reliable and
rapid method for measuring it is needed.
Few techniques for the determination of
calcium in biological fluids are sensitive, and
most are extremely tedious. The wide vari-
ation in the salivary calcium values reported
appears to be caused by unreliable tech-
niques employed or differences in sampling
procedure. Early investigators'-4 used oxa-
late-permanganate titrations; recently the
emphasis has been in complexometric titra-
tions,5 6 which have since been followed by
use of flame photometry.7' 8
In 1960 Willis9 applied atomic absorption
spectroscopy to the determination of cal-
cium in serum, and Newbrun10 later made a
preliminary investigation of the application
of Willis' method to saliva.
The present report describes a method
for the sampling of mixed saliva and a
modification of Willis' procedure to suit sal-
ivary determinations. The precision and ac-
curacy of this technique are assessed, and
the mean calcium values given for 168 in-
dividuals.
Materials and Methods
ATOMIC ABSORPTION SPECTROPHOTOME-
TER.-The atomic absorption spectropho-
tometer was assembled from commercially
available components to the specification of
Box and Walsh."
This investigation was supported by a grant from the Na-
tional Health and Medical Research Council of Australia.
Received for publication January 20, 1964.
* Present address: Department of Physiology, Medical
School, University of Sydney, Sydney, Australia.
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Two modifications of the apparatus were
made: The gelatin grating of a Hilger D290
monochromator was replaced by a quartz
prism to improve the resolution at all wave
lengths and the venturi capillary tube was
modified according to Willis.9
For routine calcium estimations, the hol-
low cathode lamp was run at 50 cycles at a
current of 10 ma. The photomultiplier was
run at 450 v. d.c. and the slit-width of the
monochromator set to give 100 per cent
transmission of the calcium light at the
maximum gain setting of the amplifier. Air
pressure was set at 12 psi and the nebulizer
adjusted to give maximum absorption while
aspirating a calcium solution. Acetylene was
used as the inflammable gas.
STANDARD SOLUTIONS.-Analytical rea-
gents were used at all times. All volumetric
glassware was cleaned in 5 N HCl for at
least 24 hours, then washed repeatedly in
distilled water prior to use. All standard
solutions were stored in polyethylene bottles.
Calcium.-A concentrated stock solution
containing 1,000 ppm calcium was prepared
by dissolution of 2.4973 gm. oven-dried cal-
cium carbonate in a minimum of dilute HCl
and made up to a volume of 1 liter. Stand-
ards of 0, 2, 4, 6, 8, and 10 ppm calcium
were made for routine use.
Strontium chloride.-A 5 per cent solution
was prepared by dissolving 152 gm. SrCl2
6H20 in distilled water and making up to
1 liter.
COLLECTION OF SALIVA.-The subjects
used in this study were child patients, stu-
dents, and staff of the United Dental Hos-
pital, Sydney. Saliva was collected 1-1 2
hours after a meal as follows: First, the sub-
ject rinsed his mouth several times with tap
water. Then, after 1 or 2 minutes to allow
clearance of the water, the subject expecto-
rated into a 20-ml. polystyrene container
for a period of 10 minutes. The same proce-
885
886 GOW
dure was adopted for the collection of stim-
ulated saliva, except that the subject chewed
paraffin wax during the 10-minute collection
period. In addition to the saliva collected as
above, 8 samples were collected under a layer
of toluene.
Results
INSTRUMENTAL FACTORS INFLUENCING THE
SENSITIVITY OF CALCIUM ABSORPTION.-The
variables which may affect the sensitivity
of the apparatus are (1) wave-length setting
and slit-width of the monochromator; (2)
composition of gases applied to the burner,
length of light-path through the flame and
region in the flame through which the light-
beam passes; and (3) effectiveness of nebu-
lization of the sample.
TABLE 1
CALCIUM CONTENT OF ACID SUPERNATANT,
ASHED AND UNASHED, AND OF CENTRIFUGED
RESIDUE IN 10-ML. SAMPLES OF SALIVA
CALCIUM
Unashed
(AG/1OO
Ashed
SAMPLE Supernatant Supernatant
1.......... 810
2.......... 630
3.......... 580
4.......... 610
5.......... 705
6.......... 585
7.......... 530
8.......... 885
9.......... 630
10 .......... 735
In this investigation a further gain in
sensitivity was made possible by placement
of a horizontal slit at the vertical entrance
slit of the monochromator. Although this
extra slit decreased the light which reached
the photomultiplier, a gain in sensitivity by
a factor of approximately 1.2 was obtained.
The sensitivity of the apparatus could be
reduced when desired by rotating the burner
and consequently reducing the length of
flame through which the beam of light
passed.
SAMPLING OF SALIVA.-Saliva, whether
collected under toluene or not and irrespec-
tive of the time between collection and sam-
pling, could not be diluted and directly
aspirated into the flame. The presence of
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J. dent. Res. September-October 1965
particulate matter and mucin clot in the
saliva caused blocking of the capillary tube
in some samples. There was also a lack of
agreement in replicate estimations from
identical volumes from many samples. Cen-
trifugation of whole saliva effectively re-
moved particulate matter but also removed
variable amounts of calcium. Estimation of
the calcium in the centrifuged mucin-debris
clot of 6 saliva samples, each 6-8 ml., gave
values of retained calcium between 36 and
318 ug.
This loss of calcium was overcome by
acidifying the sample with 10 N HClI, add-
ing an amount of the acid which correspond-
ed to 1 per cent of the total volume. In each
sample the pH of the saliva was reduced to
approximately 1.5. Centrifugation of this
acidified secretion gave a visibly homogene-
ous, slightly opalescent supernatant. An
agreement of better than 1 per cent existed
between all subsequent replicate estimations
which employed this procedure. After acidi-
fication of the saliva, the maximum calcium
retained in the mucin clot (Table 1) was
ML SALIVA)
5.5 gg. and was approximately 0.5 per cent
Residue of the total calcium of the entire sample.
Consequently, each sample was acidified
820 1.0 and centrifuged before making an estima-
630 1.0 tion.
565 1.0
610 1.0 CHEMICAL FACTORS INFLUENCING THE
705 2.0 SENSITIVITY OF CALCIUM ABSORPTION.-The
585 1.5 following factors have been considered: (1)
530 3.5 suppression of absorption by phosphate; (2)
895 5.5
640 3.5 effects of other inorganic ions (Na and K)
735 4.0 (these were demonstrated by using solutions
of calcium to which was added the variable
under observation; the effects of Mg and Cl
were also observed); and (3) effects of or-
ganic components.
Effects of phosphate.-The level of 40 ppm
(as orthophosphate) was used as the prob-
able upper physiological limit, being based
on figures reported by Afonsky.12 EDTA
(disodium ethylenediaminetetraacetate) in
concentrations of 2,000-10,000 ppm failed
to prevent the depressing effect of 40 ppm
phosphorus. Strontium chloride (2,500 ppm
Sr) also failed to suppress the effects of
phosphorus; the transmission value of 10
ppm calcium was 3 per cent less than a
phosphate-free calcium solution. When the
strontium concentration was increased to
4,000 ppm, no depressing effect due to 40
ppm phosphorus was observed.
Vol. 44, No. 5 SALIVARY CALCIUM ESTIMATION 887

Effects of other inorganic ions.- diluted saliva is aspirated into the flame of
1. Sodium and potassium. A 1-2 per cent an atomic absorption spectrophotometer
enhancement, which was dependent on the and the result checked by standard solu-
over-all sensitivity, was observed using con- tions which contain 0-10 ppm calcium (as
centrations of sodium and potassium be- CaCl2), 4,000 ppm strontium, and 100 ppm
tween 50-500 ppm and 75-300 ppm, re- sodium (as NaCl).
spectively. Over these ranges the enhance- An assessment of the accuracy of any
ment effect was constant. When the solution determination using this technique was ob-
contained 100 ppm sodium and 4,000 ppm tained from the results of recovery experi-
strontium, the presence of 125 ppm potas- ments. Duplicate 0.5- and 1-ml. portions of
sium produced no further enhancement saliva were first analyzed for calcium, then
effect. similar portions were diluted in the same
2. Magnesium. There was no observable strontium concentration to which a known
effect of 5 ppm magnesium on the absorp-
tion of either 3 or 8 ppm calcium in 4,000 TABLE 2
ppm strontium. RESULTS OF RECOVERY EXPERIMENTS
3. Chloride. The acidification of the saliva FOR CALCIUM
sample with HC1 resulted in a final chloride
ion concentration in the diluted aliquot of
approximately 450 ppm. There was no ob- CALCIUM (PPM)
1/10 Diluted Saliva
servable effect of chloride in this concentra- +Added Calcium
tion on the absorption of 5 ppm calcium. 1/10 Diluted (A) Esti- (B) Cal- A/B
Higher concentrations of chloride (viz., 0.1 SAMPLE Saliva mated culated Per Cent
M and 1 M) resulted in depression of the 6.35
absorption figure for 5 ppm calcium by 1 8.20 8.15 100.6
2 6.20 8.05 8.00 100.6
1 per cent and 7 per cent, respectively. 3 4.55 6.35 6.35 100.0
CALCIUM ABSORPTION IN THE PRESENCE 4 7.40 9.00 9.20 97.8
OF 4,000 PPM STRONTIUM.-The relationship 5 4.80 6.65 6.60 100.8
of absorption to concentration of calcium 6 4.20 6.10 6.00 101.7
7 6.10 8.00 7.90 101.3
from 0-10 ppm was found to be linear. Ac- 8 6.90 8.70 8.70 100.0
cordingly, all saliva samples were diluted in 9 5.15 6.90 6.85 100.7
a solution so as to give a finished concentra- 10 5.00 6.80 6.80 100.0
tion of 4,000 ppm Sr and then estimated 11 5.80 8.00 7.85 101.9
12 5.30 7.25 7.35 98.6
against standard calcium solutions having 13 5.60 7.65 7.65 100.0
the same Sr concentration plus an additional 14 77.35 9.55 9.40 101.6
100 ppm Na. 15 5.30 7.40 7.35 100.7
EFFECTS OF ORGANIC COMPONENTS.- 16 7.50 9.55 9.55 100.0
17 6.90 9.00 8.95 100.5
Since the preliminary sampling procedure
fails to remove all protein material from
suspension, some saliva supernatants were quantity of calcium (1.8 and 2.05 ppm in
divided into two parts. A sample (0.5 ml.) samples 1-10 and 1-17, respectively) had
was ashed, redissolved in HCl, and diluted been added. The estimated and calculated
to give a concentration of 4,000 ppm Sr, values are compared in Table 2.
while an equal quantity was diluted and The reproducibility of any one determi-
estimated directly. The results shown in nation (i.e., the precision of the technique)
Table 1 demonstrate that residual protein was assessed by a series of readings of the
matter aspirated into the flame does not
affect the amount of absorption. same standard calcium solution. Twelve sep-
TECHNIQUE RECOMMENDED FOR CALCIUM arate estimations of a 6-ppm standard re-
ESTIMATION IN SALIVA.-Saliva is collected, sulted in a standard deviation of + 1.1 per
acidified, and centrifuged. Duplicate ali- cent of the calcium value. On another occa-
quots are then pipetted from the superna- sion, at a lower sensitivity, 15 estimations
tant solution. A solution containing 4,400 of a 3-ppm standard resulted in a standard
ppm strontium (as SrCl2 6H20) is added in deviation of + 1.5 per cent.
the ratio of 9 volumes to 1 of saliva. The CALCIUM IN STIMULATED AND UNSTIMU-

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888 GOW
TABLE 3
CALCIUM CONTENT OF WHOLE SALIVA OF SUBJECTS AGED EIGHT TO FIFTY YEARS
SUBJECTS
Age
SALIVA (yr.)
f
8{14
Unstimulated
115 50
Stimulated (paraffin).. f8-14
115_50
LATED SALIVA.-In order to compare the
results obtained by this method with those
of existing investigations, the subjects were
divided into two age groups-those under
fifteen years and those fifteen years and
over. The results of analysis of the saliva
from 168 subjects are listed in Table 3.
Discussion
Direct application of the technique of
Willis9 to saliva was found to be unsatisfac-
tory, owing to the higher phosphate levels
which occur in saliva, causing depression of
the calcium absorption. Furthermore, saliva
could not be directly diluted and aspirated
into the flame, as proposed by Newbrun,10
since the salivary particulate matter result-
ed in clogging of the capillary tube.
Since many methods already exist for
salivary calcium determination and because
none of these is of unquestioned accuracy,
emphasis here has been placed on the cri-
terion of internal consistency.
The modified technique proposed in this
paper results in a precision of +1.5 per
cent of the calcium value or better. Since
the effects of the principal inorganic ions
have been shown to be negligible in the
case of K, Mg, and Cl and controllable in
the case of Na and P, it has been concluded
that this modified technique is thus sensi-
tive to the presence of calcium only. Further
evidence of the above conclusion is supplied
by the results of the recovery experiments
(Table 2), which clearly show that an added
quantity of calcium can be recovered within
the order of accuracy of the method. Rela-
tively small quantities of calcium were add-
ed to the saliva samples, since larger addi-
tions indicate a higher accuracy than would
be routinely attainable. Thus the accuracy
of the method is at least + 2 per cent.
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J. dent. Res. September-October 1965
CALCIUM
Range Mean Standard
No. (mg. per cent) (mg. per cent) Error
34 3.85-9.20 6.27 0.256
2.75-9.85 6.21 .244
34
54 3.66-0.65
3.75-9.10 5.88
5.85
.160
0.183
46
The values shown in Table 3 showed no
significant difference between stimulated
and unstimulated saliva and no difference
between the two age groups.
Examination of the mean salivary cal-
cium values reported by other investigators
reveals wide differences between them. Such
differences could have resulted from the use
of inaccurate techniques but may also have
arisen from faulty sampling of the secretion
-that the centrifugation of whole saliva
results in loss of calcium from the super-
natant was shown by Leung."3 Thus, tech-
niques3' 7, 14 which employ centrifugation of
the whole saliva prior to analysis determine
an amount which is less than the total cal-
cium.
The wide variation in the calcium values
reported by Brawley and Sedwick4 com-
pared with those of Becks15 suggests that
oxalate-permanganate techniques are not re-
liable when applied to saliva. The lower
values of this study are similar to those of
Dreisbach,8 who employed flame photome-
try.
The atomic absorption method has, as
its major advantages, sensitivity and rapid-
ity. Routine calcium estimations of whole
saliva require as little as 0.2 ml., while the
time required for estimation of duplicates
and a standard solution (to check the sen-
sitivity) is approximately 15-20 seconds.
Other features of this method over many
existing techniques are high precision, accu-
racy, and relatively greater freedom from
interference.
Summary
Atomic absorption spectroscopy was ap-
plied to the analysis of calcium in saliva
without prior ashing, using a simple instru-
ment. The saliva could be readily sampled
Vol. 44, No. 5
provided it was acidified and centrifuged to
remove particulate matter. Tenfold dilu-
tions in 4,400 ppm strontium (as SrC12*
6H20) of the acidified supernatant could be
aspirated into an air-acetylene flame and
estimated against standard calcium solu-
tions of 0-10 ppm calcium, containing the
same strontium concentration plus 100 ppm
sodium (as NaCI).
The mean calcium value of the unstimu-
lated saliva of 68 subjects was 6.24 mg. per
cent, and that for the stimulated saliva of
100 subjects was 5.87 per cent.
Differences in calcium values of both
stimulated and unstimulated saliva of two
groups of subjects varying in age (under and
over fifteen years) were not significant.
I wish to express my thanks to Dr. J. B. Willis, of
the Commonwealth Scientific Industrial Research
Organization, Melbourne, and to Dr. B. G. Davey,
of the Faculty of Agriculture, University of Sydney,
for their advice, and to Dr. K. W. Knox, Assistant
Director of the Institute of Dental Research, for en-
couragement during the course of this work.
References
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in the Whole Blood, Plasma, and Serum by Direct
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SALIVARY CALCIUM ESTIMATION 889
2. KRASNow, F., KARSHAN, M., and KREJCI, L. E. De-
termination of Calcium and Phosphorus in Saliva,
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