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The sialic acid content of human saliva
BlOCHEMlCAL
Vol. 4, No. 1,196?
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THESIALICACID CONTENTOFHUMANSAlIVA I.
5. E. Mergenhagen, and R. Xaes
Zipkin,
National Institute of Dental Research, National Inetitutee of Health, Public Health Service, U, S. Dept. of Health, Education and Welfare, Bethesda, Md. Received December29, 1960 The occurrence of sialic
acids in many mucoproteins of animal tissues
(1,2) coupled with the presence of "mucolds" in humansaliva prompted the present study.
To our knowledge no published information has demonstrated
the presence of sialic
acid in either whole or parotid humansaliva despite
the well-documented presence of this class of compoundsin the ealivary (1,2) * Using the direct sialic
Ehrlich reaction,
glands
Schneyer (3) could not detect
acid in parotid saliva, Samplesof parotid saliva (P.S.) using the Lashley cup (4) and mixed
whole saliva (W.S.) were obtained consecutively from 18 individuals.
with paraffin-gum stimulation
Por the determination of total
sfalic
acid, 1.0 ml of
saliva was hydrolyzed with 0.1 ml 3N R2S04at 80' C for 1 hour, and 0.2 ml were taken for analysis according to the thiobarbituric Warren (5) uaing N-acetylneuraminic shown in Table 1. relation
The presence of sialic stantiated
acid (NANA) as standard.
The paucity of samples, however, does
between the dieease entitieo
acid procedure of
end sialic
not
The results are permit
any cor-
acid content.
acid in parotid and mixed whole saliva was sub-
ae described below.
Seventy-five
ml of parotid and mixed whole
saliva were each hydrolyzed with l/10 volume of 3N H2S04at 80' C for 1 hour. The hydrolyzatee lyophilized
and madeup to 10.0 ml with distilled
at this juncture addition, additional
were adjusted to pH 6.0 with saturated Ba(OI?)2, centrifuged,
gave a faint
Bial and faint
direct
water.
Aliquote removed
Ehrlich test (6).
In
absorption spectra were taken of the chromophoree developed on an altquot according to Warren (5).
The remaining 9.0 ml were placed
on a Dowex 2-X8, 200-400 column in the acetate form, eluted with 1.0 M 76
BIOCHEMICAl
Vol. 4, No. 1,1961
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
Table
Total
Sialic
Acid
Content
of Human Saliva
Lng % Normal p.s.* 2.0 3.7 0.9 3.6 3.9 1.8 1.5 1.6
Young Adults w.s.**P.s./w.s. 3.2 0.63 5.8 0.64 2.6 0.35 8.6 0.41 5.1 0.77 2.4 0.75 7.3 0.21 2.7 0.59
Periodontal P.S. W.S. 1.4 2.1 2.2 5.6 2.6 4.2 0.8 2.9 2.6 4.8
2.4
4.7
1.9
Mean
* *
0.54
buffer,
pH 4.6
column
in the hydrogen
Aliquots
chromatographed
(8:2:1)
and sprayed
the mixed
phores
0.47
3.5
5.3
identical
0.66
Calculations
optical
density over
reported
which
did
indicated
that
dicating
the absence
criterion
of Warren whole
saliva
elution
to both
from in whole
parotid
sialic
(5)
acid.
saliva,
and whole
5 - 10% of the
was free. 77
sialic
NANA were
to correct
Evans
while
Warren
were
indicated (8)
has (5) has
quantitative obtained
According acid
for
in the optical
study,
saliva
in saliva. total
of the hydro-
acid,
In the present
interference
waa apparent
a peak at 532 mp
as much as a 35% decrease acid.
of the chromo-
and standard
producing
in both
the NANA
chromophores
from N-acetylneuraminic
of fucose (5),
acid
It
not derive
may cause
of NANA added
taken.
of materials
derived
a spot
with
1 and 2 of Warren
due to N-acetylneuraminic
recoveries
migrated
again
column
of 7 mg% fucose
L-fucoae
revealed
on Equations
95% of the chromophore the presence
density
and after
the absence
based
were
hydroxide
the development
of thiobarbituric
before
mesh
up in 2.0 ml water.
(7)
which
was used for spectra
50-100
ethanol-water-ammonium
of Warren
preparations
aliquot
and indicated
(5).
and taken
the reagents
spectra
samples
a Dowex 50-X12,
on Whatman #l with
and the absorption
saliva
through
lyophilized,
saliva
the absorption
lyzed
passed
form,
An additional (5)
that
(6),
with
and parotid
standard.
mixed
4.0
Rheumatoid Arthritis P.S. W.S. P.S./W.S. 6.0 1.1 0.85 2.9 6.0 0.48 3.6 4.8 0.75 2.6 3.4 0.77 2.4 5.1 0.47
Parotid saliva Whole saliva
acetate
that
Disease P.S.1W.S. G.52 0.39 0.62 0.28 0.54
in parotid
in-
to the and
BIOCHEMICAL
Vol. 4, No. 1,196l
The concentrati& saliva This
than
may be attributed salivary
The absence (3)
of sialic
in parotid
of the other
acids
saliva
from
to its
presence
glands,
of sialic
has only
procedure
of Warren
We gratefully Warren
l/28
was appreciably
acid
in oral
in parotid insensitivity
the extinction
greater
the same individual
and in oral
may be due to the relative
which
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
coefficient
whole
as shown in Table
bacteria
epithelial saliva
in mixed
(9),
1.
in secretions
debris. as reported
of the direct
by Schneyer Ehrlich
reaction
of the thiobarbituric
acid
(5).
acknowledge
and the technical
the helpful
assistance
of Miss
suggestions Eileen
of Dr.
Leonard
Varah.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Chemistry Zilliken, F., and Whitehouse, M. W. Advances in Carbohydrate l3, 237 (1958). Gottschalk, A. The Chemistry and Biology of Sialic Acids and Related Substances . Cambridge Press (1960) . Schneyer, L. H. Sot. Exp. Biol. Med., 9l, 565 (1956). Lashley, K. J. Psychol. Rev., 23, 446 (1916). Warren, L. J. Biol. Chem. E, 1971 (1959). Analysis &, Whitehouse, M. W., and Zilliken, P. Methods of Biochemical 199 (1960) Ed. D. Glick, Interscience, N. Y., N. Y. Warren, L. Nature 186, 237 (1960). Evans, P.A.D. J. Lab. Cltn. Med. 55, 381 (1960). Mergenhagen, S. E., Zipkin, I., and Varah, E. To be published.
78
Vol. 4, No. 1,196?
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
THESIALICACID CONTENTOFHUMANSAlIVA I.
5. E. Mergenhagen, and R. Xaes
Zipkin,
National Institute of Dental Research, National Inetitutee of Health, Public Health Service, U, S. Dept. of Health, Education and Welfare, Bethesda, Md. Received December29, 1960 The occurrence of sialic
acids in many mucoproteins of animal tissues
(1,2) coupled with the presence of "mucolds" in humansaliva prompted the present study.
To our knowledge no published information has demonstrated
the presence of sialic
acid in either whole or parotid humansaliva despite
the well-documented presence of this class of compoundsin the ealivary (1,2) * Using the direct sialic
Ehrlich reaction,
glands
Schneyer (3) could not detect
acid in parotid saliva, Samplesof parotid saliva (P.S.) using the Lashley cup (4) and mixed
whole saliva (W.S.) were obtained consecutively from 18 individuals.
with paraffin-gum stimulation
Por the determination of total
sfalic
acid, 1.0 ml of
saliva was hydrolyzed with 0.1 ml 3N R2S04at 80' C for 1 hour, and 0.2 ml were taken for analysis according to the thiobarbituric Warren (5) uaing N-acetylneuraminic shown in Table 1. relation
The presence of sialic stantiated
acid (NANA) as standard.
The paucity of samples, however, does
between the dieease entitieo
acid procedure of
end sialic
not
The results are permit
any cor-
acid content.
acid in parotid and mixed whole saliva was sub-
ae described below.
Seventy-five
ml of parotid and mixed whole
saliva were each hydrolyzed with l/10 volume of 3N H2S04at 80' C for 1 hour. The hydrolyzatee lyophilized
and madeup to 10.0 ml with distilled
at this juncture addition, additional
were adjusted to pH 6.0 with saturated Ba(OI?)2, centrifuged,
gave a faint
Bial and faint
direct
water.
Aliquote removed
Ehrlich test (6).
In
absorption spectra were taken of the chromophoree developed on an altquot according to Warren (5).
The remaining 9.0 ml were placed
on a Dowex 2-X8, 200-400 column in the acetate form, eluted with 1.0 M 76
BIOCHEMICAl
Vol. 4, No. 1,1961
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
Table
Total
Sialic
Acid
Content
of Human Saliva
Lng % Normal p.s.* 2.0 3.7 0.9 3.6 3.9 1.8 1.5 1.6
Young Adults w.s.**P.s./w.s. 3.2 0.63 5.8 0.64 2.6 0.35 8.6 0.41 5.1 0.77 2.4 0.75 7.3 0.21 2.7 0.59
Periodontal P.S. W.S. 1.4 2.1 2.2 5.6 2.6 4.2 0.8 2.9 2.6 4.8
2.4
4.7
1.9
Mean
* *
0.54
buffer,
pH 4.6
column
in the hydrogen
Aliquots
chromatographed
(8:2:1)
and sprayed
the mixed
phores
0.47
3.5
5.3
identical
0.66
Calculations
optical
density over
reported
which
did
indicated
that
dicating
the absence
criterion
of Warren whole
saliva
elution
to both
from in whole
parotid
sialic
(5)
acid.
saliva,
and whole
5 - 10% of the
was free. 77
sialic
NANA were
to correct
Evans
while
Warren
were
indicated (8)
has (5) has
quantitative obtained
According acid
for
in the optical
study,
saliva
in saliva. total
of the hydro-
acid,
In the present
interference
waa apparent
a peak at 532 mp
as much as a 35% decrease acid.
of the chromo-
and standard
producing
in both
the NANA
chromophores
from N-acetylneuraminic
of fucose (5),
acid
It
not derive
may cause
of NANA added
taken.
of materials
derived
a spot
with
1 and 2 of Warren
due to N-acetylneuraminic
recoveries
migrated
again
column
of 7 mg% fucose
L-fucoae
revealed
on Equations
95% of the chromophore the presence
density
and after
the absence
based
were
hydroxide
the development
of thiobarbituric
before
mesh
up in 2.0 ml water.
(7)
which
was used for spectra
50-100
ethanol-water-ammonium
of Warren
preparations
aliquot
and indicated
(5).
and taken
the reagents
spectra
samples
a Dowex 50-X12,
on Whatman #l with
and the absorption
saliva
through
lyophilized,
saliva
the absorption
lyzed
passed
form,
An additional (5)
that
(6),
with
and parotid
standard.
mixed
4.0
Rheumatoid Arthritis P.S. W.S. P.S./W.S. 6.0 1.1 0.85 2.9 6.0 0.48 3.6 4.8 0.75 2.6 3.4 0.77 2.4 5.1 0.47
Parotid saliva Whole saliva
acetate
that
Disease P.S.1W.S. G.52 0.39 0.62 0.28 0.54
in parotid
in-
to the and
BIOCHEMICAL
Vol. 4, No. 1,196l
The concentrati& saliva This
than
may be attributed salivary
The absence (3)
of sialic
in parotid
of the other
acids
saliva
from
to its
presence
glands,
of sialic
has only
procedure
of Warren
We gratefully Warren
l/28
was appreciably
acid
in oral
in parotid insensitivity
the extinction
greater
the same individual
and in oral
may be due to the relative
which
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
coefficient
whole
as shown in Table
bacteria
epithelial saliva
in mixed
(9),
1.
in secretions
debris. as reported
of the direct
by Schneyer Ehrlich
reaction
of the thiobarbituric
acid
(5).
acknowledge
and the technical
the helpful
assistance
of Miss
suggestions Eileen
of Dr.
Leonard
Varah.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Chemistry Zilliken, F., and Whitehouse, M. W. Advances in Carbohydrate l3, 237 (1958). Gottschalk, A. The Chemistry and Biology of Sialic Acids and Related Substances . Cambridge Press (1960) . Schneyer, L. H. Sot. Exp. Biol. Med., 9l, 565 (1956). Lashley, K. J. Psychol. Rev., 23, 446 (1916). Warren, L. J. Biol. Chem. E, 1971 (1959). Analysis &, Whitehouse, M. W., and Zilliken, P. Methods of Biochemical 199 (1960) Ed. D. Glick, Interscience, N. Y., N. Y. Warren, L. Nature 186, 237 (1960). Evans, P.A.D. J. Lab. Cltn. Med. 55, 381 (1960). Mergenhagen, S. E., Zipkin, I., and Varah, E. To be published.
78