Research

SALIVA VISCOSITY MEASUREMENTS

-
Y. ERICSSON, ODONT. DR., STD r>. ~T.JERNSTR(hf, rms., STO~:KFIOI,;M. Su-wmx

T HE significance in odontology
pointed out on a number of occasions.
the liquid flow but also the diffusion:
of the viscosity of the saliva has been
High viscosity influences not only
for the majority of the substances
tested, the diffusion coefficient is inversely proportional to the viscosity of the
medium (Jacobs, 1935). It has been suggested that high viscosity of the
saliva promotes carious dissolution by decreasing the rate of flow ant1 dif-
fusion, and that it is responsible, through the same mechanism, for nn in-
crease in the precipitation of calculus. Increased retention of adhesion pros-
theses has a,lso been attributed to high viscosity. Actual investigations of the
correlation between the viscosity of the saliva and the factors mentioned are,
however, few in number.
Pickerill (1912) conducted qualitative observations concerning a rela-
tionship betwen high mucin content-the viscous component of the saliva-
and high caries incidence.
MGller in 1928 examined rest saliva of 225 children 4 to 15 years of age
and found an average relative viscosity of 1.9, taking water as unity. Chil-
dren with high caries incidence showed higher average viscosity than the
caries-resistant. Miiller used the Hess viscometer, which is described later in
this article.
0stlund (1947), using Ostwald’s viscometer, determined the viscosity of
filtered saliva of 9 subjects. The more viscous of these salivas indicated no
increased prosthesis retention in a model experiment, rather the opposite
tendency suggesting itself.
Rathje and Friihlich (1949) studied the connection between caries in-
cidence and the saliva’s viscosity and rate of secretion. The viscosity was
evidently determined with a viscometer of the Ostwald type. The authors
made the following observation : “It is a familiar fact that the viscosity of
the saliva. decreases on stirring and hence during its determination lower
values will be obtained on repeating the measurements. The values stated
have been obtained from the first viscosity measurement.” Rathje and Friih-
lich found that the viscosity decreases with the rate of secretion and tha.t the
caries incidence exhibits parallelism with the viscosity of the rest saliva.

Original Investigation
The original purpose of this investigation, which was entered upon with-
out knowledge of the greater part of the literature cited, was to establish
whether there actually existed that relationship between the viscosity of the
From the Department of Operative Dentistry of the State Dental College (Head Pro-
fessor G6sta Westin).
This investigation forms part of coordinated work on dental caries sponsored by the
Swedish Medical Research Council tbpough its Subcommittee for Caries Research.
1465
saliva and the incidence 011caries which clinical observations seemed to sug-
gest Measurements were first performed with the Ostwald viscometer iOst,-
wald and Luther, 1943), which functions on the principln of a comparison l)e-
tween the time ol’ flow of the test liquid through :I vert ieal capillary a,nd t,he
time for a similar volume of water to flow through the same capillary. Cer-
tain modifications of this viscometer which aimed at preventing the forma.tion
of air buhl~les M;erc also used.

Fig. I.-Sketch of the Hess viscometer.

In the very first test it was observed that the viscosity of freshly secreted
saliva sank rapidly and uniformly. The long flow periods required for the
saliva are a distinct disadvantage of the Ostwald viscometer in cases in which
the viscosity changes rapidly during the course of the determination. In the
following experiments, therefore, a viscometer has been used which was con-
structed on the lines evolved by Hess (1906, 1907)) who, in the same articles,
described the theoretical basis of this instrument. Fig. 1. is a sketch of the
apparatus. Two horizontally mounted glass tubes each have an end section,
A and A,, 15 cm. in length and 4 mm. internal diameter, a central part 10 cm.
long and 0.5 mm. internal diameter, and the other end 30 cm. in length and 2
mm. internal diameter. These ends are connected through a three-way tap
(B) with a. small rubber balloon (C) with vent (D) . Into the end (A) there is
introduced with a pipette at least 1 ml. saliva which has been run through
a platinum strainer with 0.4 mm. holes to remove any coarser particles. Into
the tube end (A,) a corresponding amount of distilled water is pipetted. The
two liquid meniscuses are drawn in turn through the capillary to the zero marks
on the two scales E and E,. Thereafter suction is applied simultaneously to
each tube by means of the rubber balloon, until the water reaches a given
mark, say 25 cm. If the saliva in the other tube has at that moment reached the
9.7 cm. mark, then the relative viscosity is 25/9.7, provided the two tubes are
absolutely alike; otherwise, a correction factor must be found by drawing
water through both tubes simultaneously. Small variations in room tempera-
ture have little effect on this viscosity determination (Hess, 1907). After the
liquid columns have been driven back again with the balloon, the viscometer
is ready for further determinations.
 SALIVA VISCOSITY MEASUREMENTS 1467

Method
The viscometer described was used in the following experiments. The
suction pressure was between 40 and 60 cm. H,O, the flow then occupying less
than ten seconds.
With this pressure the Hagen-Poiseuille viscosity law should hold fairly
well for this apparatus. With higher pressure disturbances arise through
turbulent flow; with the lowest pressures disturbances arise through the
“structure viscosity.” This structure viscosity is due to the occurrence of
colloids or still coarser dispersed phases, especially those of fibrillar or reticu-
lar structure, which give rise to abnormally high values of the viscosity at low
pressures. Hoepfner (1943) described structural viscosity in human saliva.
Correction factors for two tubes used for saliva were calculated from 10
successive determinations with distilled water and with 25 cm. water columns
in the control tube. The results are given in Table I. All measurements were
performed at room temperature and after moistening the glass wall with the
appropriate liquid.
TABLE I
-
TUBEC TUBED
23.5 23.8
23.5 23.8
23.4 23.9
23.4 23.8
23.3 23.8
23.3 23.8
23.3 23.7
23.3 23.8
23.3 23.8
23.2 23.8
M 23.4 23.8
20.11 kO.04

The requisite amount of saliva for the determination having been col-
lected, at least half an hour after the most recent meal, the time was noted;
all measuring times were reckoned from this time. The following series of
determinations were performed :
1. Mixed rest saliva (= parotid + mandibular saliva) ; 11 tests on dif-
ferent subjects.
2. Mixed saliva, stimulated by paraffin chewing, from some of the same
subjects.
3. Mixed rest saliva, double portions divided into two, one of them be-
ing filtered through ordinary filter paper under slight pressure; simultaneous
determinations on both fractions.
4. Pure mandibular saliva (= the pooled secretion of submandibular
and sublingual glands) ; collected by suction directly at the gland orifice.
The parotid saliva was eliminated by carefully rinsing the mouth with water
before taking the sample and then isolating the parotid papillae with rubber
cups (Gore, 1938) or by tamponing firmly with cellulose wads.
5. Pure mandibular saliva ; double portions ; the viscosity development
of one of these was measured after careful mixture with one-half volume pure
parotid saliva from the same subject (collected by Gore’s rubber cups si-
1468 Y. ERICSSOK ANL, L. STJERNSTRGM

multaneously with the mandibular saliva ) ; as a cdomparison the viscosity

development of the other portion was determined after mixing water in the
same proportion. In some experiments the mandibular saliva was mixed in-
s’tead with one-half volume 1 per cent solution of malt-a-amylase.
The experiments with pure mandibular saliva and with mandibular saliva
with addition of parotid saliva or a-amylase solution were performed in view
of information from Gore (1935) and Cavalli (1940). Cavalli stated that
parotid saliva contains an enzyme which splits the mucin of mandibular saliva
and thus reduces its viscosity and raises its content of reducing substances.
Gore stated that parotid amylase has this effect. It, is well kt~wn that
amylase is the dominating enzyme of parotid saliva and practicall>- identical in
its action with cY-amylase from other sources.
6. Pure mandibular saliva, mixed with a similar volume of a suspension
of oral bacteria in physiological salt solution. The suspension was prepared in
the following way. A known volume of mixed saliva from the same person
was centrifuged, the sediment washed repeatedly with physiological salt solu-
tion, and finally suspended in half of t,he original rolume. On mixing with an
equal amount of pure mandibular saliva the concentration of cells (not, only
bacteria) was again about the same as in the mixed saliva.
The last-mentioned experiment was l)erformed as ;I result of a report of
Rogers (1948) : on incubation of saliva. N-acetglglucosamine, evidently from
the mucin, is liberated, due to the cell elements in the saliva and not, IO enzymes
in the pure secretion.
7. Pure mandibular saliva was mixed with M/I00 ascorbic acid solution
in the proportion of 2 :I.
8. 1 ml. pure mandibular saliva + 0.5 ml. M/100 ascorbic acid solution
t 1 drop 1 per cent H,O,.
9. 1.5 ml. pure mandibular saliva t 1 drop 3 per cent ILO,.
10. 1 ml. pure mandibular saliva + 0.5 ml. M/100 ascorbic acicl t 1. drop
1 per cent CUSO,.
Experiments 7-10 were performed as a result of information in the litera-
ture which stated that reducing substances, and in particular ascorbic acid,
on oxidation reduce the viscosity of different polysaccharides; more will be
said of this in the Discussion.
Results
1. Of this series, those values measured five, thirty, sixty, and one hundred
eighty minutes after secretion are given in Table II. Two typical curves are
shown in Fig. 2 where the abscissae give the times after taking samples, and the
ordinates the lengths of the saliva column: the same symbols are used in all
the subsequent figures. In each case the flow volume increased with time,
indicating a decreasing viscosity, the change occurring most rapidly immedi-
ately after taking the sample.
2. Saliva stimulated by chewing showed consistently lower initial vis-
cosity (greater flow volume) and less change than with rest saliva.
 SdLIV.4 VISCOSITY MEASUREMENTS 1469
TABLE II

SALIVA COLUM N IN CM. AFTER:

-
SUBJECT 5' 30' 60' 180'
y -.__
I.K. - 3.5 6.8 8.0 10.3
B. J. 16.0 17.7 18.7 19.1
U. J. 10.5 11.0 12.3
T. T. 11.8 12.7 14.9
H. S. 10.3 12.7 14.4 16.2
L. s. 11.0 11.1 12.3 14.4
I. H. 10.8 13.5 14.3 15.8
G. E. 13.0 15.6 16.9 18.2
E. A. H. 12.6 13.6 14.2 15.3
L. T. 8.6 10.1 11.6 13.9
G. A. 17.7 19.0 I 20.1 20.2
L

20

15

10

I I I I I I
30 GO 90 120 150 180 min.
Fig. Z.-Mixed rest saliva. Subjects G. T,;;d,‘;,K., Table II. X, After 555 min.: Y, after

3. Typical curves from simultaneous determinations of filtered and un-

filtered saliva are shown in Fig. 3. Determinations with filtered fractions
were begun somewhat later than with the unfiltered on account of the pro-
tracted filtering time. It appears that the viscous component was retained on
the filter, so that the viscosity of the filtered fraction was close to that of the
water and remained practically unchanged over several hours. The viscosity
of the unfiltered fraction decreased still further in the same period.
. 4. Pure mandibular saliva showed a varying viscosity development, as
can be seen from the three typical curves in Fig. 4. Results of type 1 or 2,
i.e., slow or no decrease in viscosity, were most common in the experiments
performed.
1470 Y. ERICSSON AND 1,. STJERNSTRGM

25 :m.

. ; ....... ..................
x

20

15

10

I 8 , I
30 GO 90 120 150 min.
“ig. 3-The effect of Altering on the viscosity of the saliva. Upper curve. flltered; lower
curve, unflltered. X and Y = after twenty-four hours.

Fig. 4.-Pure mandibular saliva from 3 subjects.

 SALIVA VISCOSITY MEASUREMENTS 1471

5. Neither parotid saliva nor amylase solution had any apparent effect on
the viscosity development in the mandibular saliva, as is seen from t,he t.ypical
curves in Figs. 5 and 6.
6. Suspensions of oral bacteria and other cell elements of the saliva had
no obvious influence on the viscosity development; see curves in Fig. 7.
7. Ascorbic acid alone had no definite effect on the viscosity development ;
curves of the same type as in Fig. 7.

cm.
2. . .
,. *L-r,--, _____- * ______--_------- t nm_--------m---- - ------ -4

0' 1
30 610 $0 120 150 180 min.
Wig. 5.-Mandibular saliva. extraorally mixed with parotid saliva (---------). compared with the
same mandibular saliva. mixed with water in the sme proportion (-).

Fig. 6.-Mandibular saliva, mixed with a-amylase solution (o), compared with the same mandib-
ular sahva, mixed with water in the same proportion. ( l ).

b
30 60 90 min.
Fig. ?.-Mandibular saliva plus suspension of saliva cells (- - - - -). Comparigon : the same
saliva plus water ( -). X and Y = after 1,005 minutes.

8. Ascorbic acid plus hydrogen peroxide strongly reduced the viscosity ;

typical curves in Fig. 8.
9. Hydrogen peroxide alone did not affect the viscosity; curves of the
same type as in Fig. 7.
10. Ascorbic acid plus copper ions considerably reduced the viscosity. In
several cases the reduction in viscosity occurred almost instantaneously. See
1472 Y. ERICSSON ANI) I,. STJERKSTRGM

Fig. 9 where the measurements were first performed after addition of’ ascorbic
acid alone, and again fire minutes later after addition of 1 drop of the col)-
per sulfate solution,

m.

2( :m.

1:

l(

5
30 60 90 min. 30 60 90min.
Fig. 8. Fig. 9.
Fig. 8.-Mandibular saliva plus ascorbic acid plus HZOY ( l ). Comparison: the same saliva
plus water (0).
Fig. 9.-Mandibular saliva plus ascorbic acid solution. Dashecl line marks the addition
of 1 drop of 1 per cent copper sulfate solution.

Discussion
In addition to the authors mentioned (Cavalli and Rathje and E’rGhlich)
and ourselves, reduction in the viscosity of the saliva has been observed by
Rogers (personal communication, 1951) . It can, furthermore, be read from
the curves of Hoepfner (Zoc. cit.), but is not mentioned by this author.
All evidence points to the fact that the reduction in viscosity is due to
splitting of the mucin. The chemical constitution of this glyeoproteid is not
clear, but it has been confirmed that it contains considerable quantities of
acetylglucosamine (Blix, Oldfeldt: and Karlberg, 1935; Blix, 1936). Rogers
(1948) showed that N-acetylglucosamine accumulates in incubated saliva if
the further cleavage of the compound by the bacteria is prevented. The same
author has thus shown that streptococci consume N-acetylglucosamine with
the formation of ammonia, lactate, and volatile acid. It is interesting to com-
pare Rogers’s result,s with the observations of Clark and Carter (1927), that
 SALIVA VISCOSITY MEASUREMENTS 1473

both carbon dioxide and ammonia are formed in saliva maintained at room
temperature, according to these authors, by enzymatic action and not through
the bacteria of the saliva. It is also worth observing that Clark and Carter
found the most pronounced development of carbonic acid in the most viscous
salivas and then during the first two hours after taking the samples. If the
development of carbonic acid is due to further splitting of liberated acetyl-
glucosamine, this agrees well with the rapid initial reduction of viscosity in
mucin-rich saliva.
Robertson, Ropes, and Bauer (1939, 1941) found that ascorbic acid on
oxidation splits mucins of both epithelial and mesothelial origin, chondroitin
sulfuric acid, pectins and starch, all substances containing polysaccharide
groups. The presence of cupric ions seems to bring about auto-oxidation of
the ascorbic acid with simultaneous formation of hydrogen peroxide. Robert-
son and collaborators found the presence of hydrogen peroxide necessary for
the effect mentioned since ascorbic acid plus CLI++ had no effect on mucin in
the presence of catalase which destrops the hydrogen peroxide on its forma-
tion. Completely oxidized ascorbic acid had no effect either. Skanse ancl
Sundblad (1943) found the same splitting effect of ascorbic acid plus H,O, on
hyaluronic acid, heparin, and cellulose. These authors found a positive effect
also with ascorbic acid alone, as with hydrogen peroxide and other oxidizing
agents alone; the inhibitory effect of catalase they coulcl not bear out. Hale
(1944) confirmed that reducing agent,s in the presence of molecular oxygen
decreased the viscosity of hyaluronic acid and polysaccharides and that this
effect was greatly increased by Cu++ions.
As far as the original saliva is concerned the results offered here confirm
the effect of ascorbic acid in the presence of hydrogen peroxide or cupric
ions. A clinical significance of this is not excluded since vitamin C is known
to occur in the saliva (Stuteville, 1935 ; Zimmet and Dubois-Ferriere, 1937).
Concerning a possible effect of some substance in the parotid saliva, greater
uncertainty prevails. It is, however, to be noted that none of the mixed salivas
examined but several mandibular salivas showed stable viscosity.
The investigation shows that determinations of the viscosity of saliva are
of little value unless exact times between secretion and measurement are
stated.
It is also of interest to compare the saliva viscosity change and mucin
cleavage with the changes in the saliva content of the group antigens. The
mandibular saliva of most human beings shows high A and B contents in par-
ticular (Hartmann, 1941). Parotid saliva is practically free, at least from A
which is secreted by the mucous cells of the mandibular glands, and which
is precipitated on preparation together with the mucin, having a similar chem-
ical composition to it (Rex-Kiss, 1943). The ant,igen titer decreases rapidly in
a saliva sample, and after twenty hours’ incubation it has as a rule com-
pletely disappeared (Schiff and Weiler, 1931). The parallel with salivary
mucin is obvious. A-antigen from other sources (ox and pig stomachs) has
further been shown to contain a considerable quantity of acetylglucosamine
and has, on enzymatic splitting, a st,rongly decreased viscosity (Jorpes and
Thaning, 1945 ; Morgan, 1946).
1474 I’. ERICSSOK AXI) I,. ST.JERNS’l’RijM

Summary
The viscosity of the saliva was investigated using ;I Iless viscometer. The
viscosity of mixed saliva regularly sank rapidly after secretion. I’LIT~ man-
dibular saliva often showed greater stability. No certain influence on the vis-
cosity was found with cu-amylase or suspensions of salivary bacteria. The
viscosity was rapidly reduced by ascorbic acid in the presence of hydrogen
peroxide or traces of cupric ions. Parallels are pointed out with the viscosity
reduction of various polysaccharides ant1 with the disappearance of the group
antigens of the saliva.
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Blix, G., Oldfeldt! C. O., and Karlberg, 0.: Zur Chemie der AIucine und Jlucoide (Vor-
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Cavalli, G.: Der Parotisspeichel enthslt ein Ferment, das das Mucin der Submaxillaris
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Skanse, B., and Sundblad, L.: Oxidative Breakdown of Hyaluronic and Chondroitin
Sulphuric Acid, Acta physiol. Seandinav. 6: 37, 1943.
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de la salive chez l’homme eelon I’age, Compt. rend. Sot. de Biol. 124: 103,. 1937.
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