Cohen, M.

De Kock, A. C.

Retief, D. H.

English

Wits Institutional Repository on DSPACE

S. Afr. J. med. Sci. ( 1969). 34, 105-112.
THE DETERMINATION OF PHOSPHORUS IN SALIVA AND DENTINE
by
D. H. RETIEF*, A. C. DE KOCK* AND M. COHENf
* Joint Dental Research Unit o f  the University o f  the Witwatersrancl anti the Medical Research Council,
Johannesburg.
t  Department o f  Statistics, University o f  the Witwatersrand, Johannesburg.
[Received 21st J uly, 1969]
Numerous methods have been developed for the determination of phosphorus 
in biological material. Of the methods described, the most sensitive arc the colori­
metric procedures the basis of which is the formation of a phosphomolybdate complex 
which produces a blue colour on reduction.
The presence of protein in biological material may cause interference with the 
spectrophotometric methods. Thus, some of the methods used for the quantitative 
determination of phosphorus in biological material need to be modified when used 
for the determination of phosphorus in saliva and mineralized dental tissues [Jenkins, 
1966]. Of the mineralized dental tissues, dentine was selected for analysis because 
of its high protein content. Four of the accepted spectrophotometric methods for 
the quantitative determination of phosphorus were employed to determine the 
phosphorus content of saliva and dentine, modified where necessary and the results 
evaluated. In addition the sensitivity of each of the methods was determined.
The four methods employed were those of Fiskc and Subbarow [1925], Chen ct al. 
[1956], a modification* introduced by Chen el al. in their original [1956] method, 
and that of Eastoe [1965], In these methods various reducing agents and different 
ways of developing the colour are used and the spectrophotometric analysis is done 
at a wavelength most suitable for the method (Table I).
TABLE I
The reducing agent, colour development and wavelength o f  the four methods.
Method Reducing Agent
Colour
Development
Wave Length 
inn
Eiske . . . . Aminonaphtholsulfonic 
acid and sulfite
5 minutes at 
room temperature 660
Chen . . . . Ascorbic acid 2 hours at 37°C 820
“ Modified Chen” . Ascorbic acid 2 hours at 37°C 820
Eastoe . . . . Aminonaphtholsulfonic 
acid and sulfite
10 minutes at 
boiling point
815
METHODS
A Unicam SP 500 photoelectric spectrophotometer was used in this investigation.
* The modification introduced by Chen et al. in their original method consists o f the use o f reduced 
quantities of TCA filtrate and reagent in order to measure smaller quantities o f phosphorus. In 
the present paper this variation will be designated the “ Modified Chen” method.
106 The South African Journal o f Medical Sciences
Sensitivity
A comparison was made of the sensitivity of the four methods. This was done by 
recording the absorbance of standard phosphate solutions containing from 0.10- 
10,ig of phosphorus. Standard curves were constructed for each method by plotting 
the phosphorus content against the absorbance (Pig. 1).
Saliva analysis
Because we were not concerned with the factors that control the composition of 
saliva [Jenkins, 1966J stimulated saliva was collected by chewing rubber tubing 
and pooled for this comparative study. The saliva was centrifuged at 3,000 revolutions 
per minute for ten minutes to remove the epithelial cells and suspended matter. 
Although it has been reported that centrifugation lowers the phosphorus content of 
saliva [Wainwright, 1938] we found that the suspended matter interfered with the 
subsequent analytical techniques if not removed. The centrifuged saliva was filtered 
through Whatman No. 1 filter paper and the filtrate utilized for the phosphorus 
determinations.
According to Becks [1938] the various phosphorus fractions of saliva can be deter­
mined as follows:
l ildalc of 
centrifuged saliva
Protein precipitated 
with \0"/, re A
Direct wet 
ashing
Total phosphorus
sResidue1
Ashing
Organic
acid-insoluble
phosphorus
^  Filtrate
Direct Direct 
analysis /  \  wet ashing
Total
Inorganic acid-soluble 
phosphorus phosphorus
The organic acid-soluble phosphorus is determined indirectly by the following 
calculation:
Organic acid-soluble P =  Total acid-soluble P — Inorganic P 
The total phosphorus and the inorganic phosphorus of saliva were determined in 
this comparative study. In both instances the protein of the saliva was removed 
prior to analysis by means of an ashing procedure in the ease of the total phosphorus 
and by precipitation with trichloracetic acid in the case of the inorganic phosphorus.
Total phosphorus o f saliva
The protein of the centrifuged salivary filtrate was removed by wet-ashing. One 
ml. of the filtrate was heated in a crucible on a furnace with 8 drops of concentrated
Relief cl ill.: Determination o f phosphorus in saliva and dentine 107
sulphuric acid until white sulphur trioxidc fumes appeared, 0.5 ml. of 30% hydrogen 
peroxide (ICO..) was then added and heated again until all the hydrogen peroxide 
was driven oil'. If the remaining fluid was still discoloured, a few drops of 30% H20 , 
were added and the procedure repeated until a clear liquid was obtained. The liquid 
was transferred to a 25 ml. volumetric flask, washed repeatedly and made up to the 
mark. Different volumes of this solution were taken for subsequent analysis.
The following volumes gave optimum recordings for each method (based on a 
salivary phosphate of J- 10 mg. %).
Method
Volume diluted 
solution (nil.)
Equivalent volume 
saliva (ml.)
Hske . . . . 5.0 0. 2
Chen . . . . 0.5 0.02
“ Modified Chen” . 0.1 0.004
tiasloc . . . . 0.5 0.02
Inorganic phosphorus o f saliva
10% Trichloracetic acid (TCA) solution was added to the filtrate of the centrifuged 
saliva to precipitate the protein. 9.5 ml. of TCA solution was added to each 0.5 ml. 
of saliva. The mixture was shaken well, allowed to stand for approximately ten 
minutes and filtered through a Whatman No. 2 filter paper. The analysis was carried 
out directly on the filtrate.
Because of the difference in sensitivity of the four methods, different volumes of 
TCA filtrate were used for each method. For saliva containing -f 10 mg. % of phos­
phorus, the following volumes of TCA filtrate gave optimum readings on the spectro­
photometer.
Method
Volume TCA 
filtrate (ml.)
Equivalent volume 
saliva (ml.)
riskc . . . . 5.0 0.25
Chen . . . . 0.5 0.025
"Modified Chen’’ . 0.1 0.005
liastoc . . . . 0.5 0.025
With the Fiskc method a further.precipitate occurred when the molybatc reagent 
was added to the TCA filtrate. This did not take place when the saliva was ashed. 
This is in accordance with Wainwright’s [1938] observation. Me thought that this 
precipitate was due to incomplete precipitation of the salivary protein with 10% 
TCA. Whenever this precipitate was formed, it was removed by further centrifugation 
at 3,000 r.p.m. for five minutes. The absorbance of the supernatant solution was 
recorded.
Dentine analysis
Sound freshly extracted human teeth were used. The teeth were dried in an oven, 
the ccmcntum removed by scraping, the teeth split and the dentine separated. To 
ensure that no enamel was removed with the dentine, only the dentine not in close 
contact with the enamel was removed. The dentine was ground to a line powder 
in an agate mortar and dried to constant weight at 105°C. The dentine was then 
dissolved either in 1 N hydrochloric acid or 0.8 M TCA, or ashed. The dentine
108 '1 he South African Journal o f Medical Sciences
dissolved in 0.8 M TCA was analysed by the Eastoc method only since this is the 
concentration of TCA used in his method.
Dentine dissolved in 1 N MCI
About 10 mg. of dentine was accurately weighed and transferred to a 250 ml. 
volumetric flask. The transfer was completed with 10 ml. 1 N HC1. The flask was 
kept at 37°C overnight, allowed to cool and made up to the mark. The undissolvcd 
protein was removed by filtration. It was found that the following volumes of the 
diluted solution gave optimum recordings with the spectrophotometer.
Method
Volume diluted 
solution (ml.)
Equivalent weight 
o f dentine (mg.)
Fiskc . . . . 5.0 0.2
Chen . . . . 0.5 0.02
"Modified Chen” . 0.2 0.008
Eastoc . . . . 0.5 0.02
Ashed dentine
A dry-ashing technique was used. Approximately 10 mg. dentine was weighed 
accurately in a crucible, 5 ml. 50% hydrochloric acid and 0.5 ml. 30% hydrogen 
peroxide (H20 2) added and gradually heated on a furnace until dry. The crucible 
was allowed to cool and the residue dissolved in 5 ml. 1 N HC1 containing 0.3% 
H20 2, facilitated by gentle heating. The solution was transferred to a 250 ml. volu­
metric flask with repeated washings and made up to the mark. The same volumes of 
this diluted solution were used with the four methods as for the dentine dissolved 
in 1 N HC1.
Dentine dissolved in 0.8 M TCA
Approximately 10 mg. of dentine was weighed accurately in a 10 ml. centrifuge 
tube and 5 ml. of 0.8 M TCA added. The tube was tightly stoppered and left in an 
oven at 37°C overnight. After cooling it was centrifuged to remove undissolvcd 
protein and 20 /xl (0.02 ml. of the supernatant fluid (equivalent to 0.04 mg. dentine) 
was used for analysis by the Eastoc method.
RESULTS
Sensitivity
From the results obtained a standard curve for each method was constructed 
(Fig. 1). Each point on the graph represents the average of four recordings.
By comparing the absorbance obtained with each of the four methods on solutions 
containing 1 /xg. of phosphorus in the test solution the sensitivity of the respective 
methods can be compared.
Method Absorbance
Fiskc . . . . 0.01 1
Chen . . . . 0.10 ( For 1 |ig. of P
“ Modified Chen” . 0.26 f  in test solution
Eastoc . . . . 0.08 J
Relief et ah: Determination o f phosphorus in saliva and dentine 109
Fit;. / Standard curves.
From these results it is clear that the Fiske method is the least sensitive and the 
“modified Chen” method the most sensitive.
Saliva analysis
Total phosphorus-ashed saliva.—The results arc given in Table 11 A.
Inorganic phosphorus-protein precipitated saliva. The results arc given in Table I IB.
TABLE It
Phosphorus in saliva, total anil inorganic
Method Mean* Standard error
Approximate 95% 
confidence interval 
for true mean
A. Total phosphorus o f  saliva
F i s k e ......................... 15.16 0.02 15.12, 15.20
C h e n ......................... 15.54 0.02 15.50, 15.58
“ Modified Chen” 15.38 0.02 15.34, 15.42
Eastoc . . . . 15.12 0.01 15.10, 15.14
B. Inorganic phosphorus o f  saliva
F i s k e ......................... 14.76 0.03 14.70, 14.82
C h e n ......................... 15.42 0.01 15.40, 15.44
“ Modified Chen” 15.38 0.02 15.34, 15.42
Eastoc . . . . 15.29 0.02 15.25, 15.33
All results expressed in mg%.
* The mean was obtained from the result of 50 analyses with each method.
Dentine analysis
The phosphorus is expressed as a percentage of the weight of dentine dried to a 
constant weight at 105°C.
Dentine dissolved in 1 N IICI.—The results arc given in Table 111 A.
Ashed dentine.—The results arc given in Table IIIB.
Dentine dissolved in 0.8 M TCA.—The results arc given in Table 111C.
no Tlw South African Journal o f Medical Sciences
T A B U ; 111
Percentage phosphorus in dentine, dissolved in IICI or TCA, or in ashed dentine.
Method Mean* ■ Standard error
Approximate 95 /,', 
confidence interval
1.... Tor true mean
A. Percentage phosphari 
F i s k c .........................
is in dentine dissoh
13.41 |
eil in N IICI 
0.02 13.37, 13.45
C h e n ......................... 13.46 i 0.02 13.42, 13.50
“ Modified Chen” 13.40 0.02 13.36, 13.44
Eastoc . . . . 13.16 ! 0.03 13.10. 13.22
B. Percentage phosphorus in ashed dentine 
F i s k c ......................... | 13.16 0.02 13.12, 13.20
C h e n ......................... 12.97 ' 0.02 12.93, 13.01
“ Modified Chen” 13.43 i 0.03 13.37, 13.49.
Eastoc . . . . 13.09 ; 0.02 13.05, 13.13
C. Percentage phosphorus in dentine dissoh 
F i s k c ......................... |
C({ in 0.8 M TCA
—
C h e n ......................... __
“ Modified Chen” —
Eastoc . . . . 12.95 1 0.02 12.91. 12.99
All results expressed is percentage of eliy weight of dentine heated to 105 C.
* The mean was obtained from the results of 50 analyses with each method.
DISCUSSION
Sensitivity
From the results obtained it is clear that the Chen, the “modified Chen" and the 
Eastoc methods arc the only ones that can be used if small amounts of saliva or 
dentine arc available for analysis. Of these methods the “modified Chen" method is 
by far the most sensitive and a single analysis can readily be made on 0.005 ml. of 
saliva or 0.008 mg. of dentine.
Saliva analysis
In the direct analysis of the protein precipitated saliva (inorganic phosphorus), 
the mean obtained with the Fiskc method is lower than that obtained with the other 
methods. There is statistical evidence that the true mean of the first population 
(Fiskc) is lower than the average of the true means of the remaining three populations. 
This is in accordance with the findings of Wainwright [1938] and others that the 
phosphorus content of saliva obtained with the Fiskc method is on the low side. 
This can partly be explained by the fact that a further precipitate was formed when the 
molybdate reagent was added to the filtrate of the protein precipitated saliva with the 
Fiskc method. This precipitate had to be removed by centrifugation prior to spcctro- 
photomclric analysis.
For the Fiske and Chen methods higher phosphorus values were obtained for the 
ashed saliva than the protein precipitated saliva. This is as expected as the inorganic 
phosphorus of saliva is approximately 95% of the total phosphorus content [Becks. 
1938]. When the “modified Chen” method was used the same results were obtained 
for the phosphorus content of the protein precipitated saliva and the ashed saliva. 
One would expect the total phosphorus content of the ashed saliva to have been 
slightly higher.
With the Eastoc method a slightly lower value was obtained for the total phosphorus 
as compared with the inorganic phosphorus of saliva. Easloe’s method, however, 
is not specific for inorganic phosphorus in the presence of organic phosphorus. The 
organic phosphate present in the saliva will be hydrolysed by the 0.34 N sulphuric 
acid of the reagent at the temperature at which the colour is developed (100°C).
Dentine analysis
The results obtained on the ashed dentine are generally lower than those obtained 
on the dentine dissolved in 1 N HCI. This may be due to the loss of some material 
during the ashing procedure.
Although the results obtained with Eastoe’s method arc slightly lower than those 
obtained with the other methods a good correlation is evident between the results 
obtained with this method.
All three methods of bringing the dentine in solution seem to be acceptable. 
Stability o f the reagents
The stability of the colour that is developed and the stability of the reducing agents 
used in the dilfcrent methods vary as shown in Table IV.
TAHLE IV
Relief et ah: Determination o f phosphorus in saliva and dentine 111
Tlte stability o f  the colour developed and o f  the reducing agents with the four methods.
Method Colour stability Stability of reducing agent
F i s k e ......................... Increases steadily for several 
several weeks
Four weeks
C h e n ......................... Stable for two hours Fresh daily
“ Modified Chen" Stable for two hours Fresh daily
Eastoc . . . . Stable for one week Fresh weekly
The colour stability is the greatest with the Eastoc method and the reducing agent 
the most stable in the Fiske method.
SUMMARY
An accurate method for the determination of phosphorus in saliva and mineralized 
tissue is often required.
A variety of methods have been described for the determination of phosphorus 
in biological samples. The most sensitive of these arc based on the reduction of a 
phosphomolybdatc complex which gives rise to a colour reaction. Four of these 
methods, in which different reducing agents and different methods of developing 
the colour arc employed, were used to determine the phosphorus content of saliva 
and dentine. In addition the same procedure was adopted for the analysis of wet- 
ashed samples.
850 phosphate determinations were carried out on saliva and dentine, using the 
methods of Fiske and Subbarow, Chen et ah, “modified Chen et ah”, and Eastoc. 
Statistical evaluation of the results showed that all four methods are accurate.
A comparison made of the sensitivity of the four methods indicates that the 
“modified Chen” method is the most sensitive.
112 The South African Journal o f Medical Sciences
REFERENCES
Becks, H. (1938). Human Saliva. IV Critical discussion of former studies of the distribution of 
salivary phosphorus fractions. J. dent. Res., 17, 217-234.
C hen, P. S., Toriuaka, T. Y. and Warner, H. (1956). Microdetermination of phosphorus. Anal. 
Chew., 28. 1756-1758.
Eastoe, J. E. (1965). Methods for the determination of phosphate, calcium and protein in small 
portions of mineralized tissues. In: Proceedings of the Second European Symposium on Calcified 
Tissues. Edited by L. J. Richcllc and M. J. Dallcmagnc. p. 265-274 (University Press, Liege).
F iske, C. H. and Suuharow, Y. (1925). Colorimetric determination of phosphorus. J. Rial. Chew., 
66, 375-400.
Jenkins, G. N. (1966). The Physiology o f  the Mouth. 3rd cd. pp. 288-316. (Blackwell, Oxford).
Wainwricjht, W. W. (1938). Human Saliva. V. A technical procedure for phosphorus analysis. 
J. dent. Res., 17, 235-250.


The determination of phosphorus in saliva and dentine

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The determination of phosphorus in saliva and dentine

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