Original Article

Preliminary Proton Nuclear Magnetic Resonance Studies of Human Saliva

A t u k o Y. N O S A K A , M.Sc.,Ph.D., Shigeru F U K U T O M I , D.D.S., S h u s a b u r o U E M U R A , D.D.S., Ph.D., Yuuji K O B A Y A S H I , M.Sc.,Ph.D.* and Y o s h i m a s a K Y O G O K U , M.Sc.,Ph.D.* Department of Oral Radiology, School of Dentistry, Tokushima University, Tokushima, Japan (Director : Prof. Shusaburo UEMURA) *Institute for Protein Research, Osaka University, Suits, Osaka, Japan (Received : Sept. 1. 1990. Revision received : May 23, 1991, Accepted : May 25, 1991)

Key Words : Proton NMR, Human parotid saliva, Human submandibular saliva

Summary-Proton NMR spectra of gustatory stimulated healthy human whole, parotid and submandibutar and sublingual saliva were measured. The typical patterns of their spectra were obtained. Among these three kinds of fluids the parotid saliva which contains preferentially serous saliva presented a relatively well resolved spectrum with satisfactory signal to noise ratio in a given short time(30

min.). For eight subjects of parotid saliva collected in the afternoon the marked increase in signal intensity in the methyl and methylene proton region of proteins(peptides)was observed as compared with those collected in the morning, reflecting the circadian rhythms in the protein concentration in saliva. On the other hand the methyl peak of lactic acid presented the opposite tendency, which might be also correlated with the circadian rhythms of the glucose metabolism of the parotid gland. The proton NMR spectra of three patients suspected of salivary gland disease were different from those of healthy subjects.

Oral Radiol. Vol.7 No.1 1991(7~11)

Introduction

b e c a u s e s e v e r a l s t e p s of p r e t r e a t m e n t

N u c l e a r m a g n e t i c r e s o n a n c e (NMR) h a s

blood are necessary.

of

T h e diffusible compo-

p r o v e d v e r y useful for b i o c h e m i c a l analysis.

nents of b l o o d and s a l i v a a r e c o n s i d e r e d to be

H i g h r e s o l u t i o n N M R s p e c t r o s c o p y h a s been

b a s i c a l l y similar.

used e x t e n s i v e l y for b l o o d a n a l y s i s 1,2), b u t the

c a v i t y (whole saliva) is a m i x t u r e of t h e

method

s e c r e t i o n s f r o m different s a l i v a r y g l a n d s and

involved

is

rather

complicated

But s a l i v a in the o r a l

also contains leukocytes, desquamated epith-

(trimethylsilyl) propionate) was used as an

elium, oral bacteria, etc., so only a few

external standard of chemical shifts and sig-

attempts have been made to examine its N M R spectrum 3-5). Saliva collected ase-

nal

intensities.

The

HDO

signal

was

ptically from the parotid, submandibular or

presaturated for 3 seconds prior to signal acquisition to suppress the water peak.

sublingual gland should not contain other

Unless otherwise stated, 320 transients were

components.

collected.

Therefore, in the present study

we examined the N M R spectra of samples of

In the same way, parotid and subman-

saliva collected in this aseptic way with a view to using this technique for physiological

dibular saliva of three different patients, who

studies on the mouth.

TSP

Materials and Methods Saliva was collected from 8 healthy men ranging age from 26 to 42 years.

Parotid

saliva was collected through a Teflon tube inserted into the orifice of Stensen's duct by stimulating with acid.

Specimens of subt

mandibular plus sublinguaI saliva were col-

I

. . . .

]

4.0

'

'

I

'

. . . .

I

' ' ' '

I

i.o

....

I

2.o PPM

lected through the orifice of Wharton's duct in the same way. About 2ml of saliva was

, . . . . . .

"

11o

,,

,

'

i

o'.o

,

collected from each subject in 10 to 30 minutes. There was no significant difference between the N M R spectra of the first and second 1 ml of the samples collected.

The

saliva was kept at 5~ for two days before N M R measurements.

' I

T h e pH of the saliva

' 4'

' ' .I

3.0I

' r 0' ' I

' ' ' I

' I

21.0

'

. . . . .

I '

' . 11.0 . . . .

I ''''olol'

. . . . . .

L'

PPM

measured just after its collection was 6.9+ 0. 6 in the morning and 7.4+0.6 in the afternoon. N M R measurements were carried out in a 5mm OD N M R sample tube with a Bruker WM-360 F T spectrometer operated in the Fourier transform mode at 360 MHz.

Spec-

tra were acquired with a sweep width of 4000Hz at 8k data points and with a relaxation delay of 3 seconds. The time domain data were apodized by an exponential window of 1Hz line broadening prior to Fourier transformation.

As a spectrometer lock, 0.

lml of D20 (99.5%) was added to 0.4ml of samples. made 8

at

All

NMR

room

measurements

temperature.

TSP

were (3-

'

[

. . . .

4.0

I

,

.... 3'.'O

' ' l

'

'

'

'

I

. . . .

2.0 PPM

]

"

,'.0

''

'

i

r

o'.0

Fig. 1 High field region of proton NMR spectra-of human saliva measured at 360MHz at room temperature. (a) whole saliva (b) submandibular plus sublingual saliva and (c) parotid saliva. Chemical shifts are shown in ppm from the signal of external TSP. 320 transients were collected in about 20 rain.

were suspected of sialoadenitis, were mea-

parotid saliva gave an NMR spectrum with

sured.

the most satisfactory signal-to-noise ratio. The spectrum shown in Fig. 1 (c) is com-

Results and Discussion

plicated because of the presence of various

Fig. 1 shows the high field regions of

species of molecules in the fluid.

typical proton NMR spectra of (a) whole

Most of the

relatively broad signals appearing at about 1

saliva (saliva pooled in the oral cavity), (b)

and 1.5-2.5ppm were attributed to methyl and

submandibular plus sublingual saliva and (c)

methylene protons of proteins (or peptides).

parotid saliva.

The sharp doublet (at 1.32 ppm with splitting

these

three

Clear differences are seen in

proton spectra.

The

of 7 Hz) has been assigned to the methyl

proton

protons of lactic acid. 5)

NMR spectra of whole saliva, and submandibular plus sublingual saliva were broad and

Fig. 3 shows the proton NMR spectra of

not well resolved, while that of parotid saliva

parotid saliva collected in the morning (about

was well resolved with a sufficient signal-to-

10 a.m.) and in the afternoon (around 3 p.m.).

noise ratio in the 30 minutes of measurement

The intensities of most of the signals in the

time.

Parotid saliva is serous, whereas the

spectrum were greater in the saliva collected

submandibular plus sublingual saliva, and

in the afternoon, the total peak area being

whole saliva are mixtures of serous and mucous saliva.

TSP

As ,mucous saliva is more

t

viscous, the broad spectra observed in Fig. 1 (a) and (b) were probably attributable to the high viscosity of mucous saliva.

As shown

in Fig. 2, after accumulation of 8000 transients (about 9 hrs.) submandibular and sublingual saliva gave a spectrum that was similar to that of parotid saliva but with rather

'

'

I

'

'

'

I

. . . .

4.0

I

. . . .

I

. . . .

3.0

I

. . . .

I

. . . .

2.0

broader peaks, indicating that it contained

I

. . . .

I

. . . .

1.0

I

'

'

O.0

P PM

similar components to those of parotid saliva but at lower concentrations.

The proton

NMR spectrum of whole saliva also gave a similar spectrum.

These results indicated

that within a short fixed measurement time

I ' l ' ' I I

4.0

' ' 1 ' ' ' 1

4.0

. . . .

[ . . . .

3.0

[

. . . .

I

. . . .

2,0 pPM

I ' ' 1 ' 1 ' ' ' ' 1

1.0

. . . .

l ' '

O.0

Fig. 2 High field region of proton NMRspectrum of human submandibular plus sublingual saliva measured at 360MHz at room temperature. 8000 transients were collected in about 9 hrs.

. . . .

I . . . .

3.0

I

. . . .

I

. . . .

2.0 PPM

I

. . . .

1 1 ' ' ' ' 1 .

0

. . . .

{

II

0.0

Fig. 3 High field region of the proton NMR spectra of human parotid saliva measured at 360MHz at room temperature. (a) Parotid saliva was collected in the morning (about 10 a.m.) and (b) in the afternoon (about 3 p m.). Chemical shifts and signal intensities are shown relative to that of TSP. 320 transients were collected in about 20 min. 9

68+- 19% 8 subjects more than that of samples collected in the morning. This peak area

increases in the afternoon.

Dawes et al.

was measured relative to that of external

parotid saliva and reported that the concen-

TSP.

This result suggests that the protein

tration of protein in stimulated parotid saliva

(and peptides) concentration of parotid saliva

showed a significant circadian rhythm being

measured the protein concentration of human

lowest early in the morning (about 2 a.m.) and highest in t h e afternoon (about 2 p.m.)2 -8~ The present results are consistent with their reports. On the contrary, the signal intensity of the methyl group of the lactic acid of parotid saliva collected in the afternoon was 46 + 20% 8 subjects less than that of saliva collected in the morning. "

'

'

I

. . . . . . . . .

4.0

I

. . . .

'

. . . .

3.0

I

. . . . . . . . .

I

2.0

Lactic acid is produced during

. . . . . . .

1.0

PPM

glucose metabolism when the parotid gland is activated to secrete saliva.

Therefore, this

difference might reflect a circadian rhythm of b

glucose metabolism in the parotid gland.

"

The pH of parotid saliva collected in the

l

morning was 6.9+_0.6, while that of saliva collected in the afternoon was 7.4_+0.6.

The

lower lactic acid concentration in the afternoon might explain the higher pH of the saliva at this time. '

'

9

I

. . . . . . . . .

4.0

I

.

.

.

.

.

.

.

.

.

3.0

.

.| .

.

.

.

.

.

.

2.0

f

1.0

PPM

The variations in composition of the saliva at different times of the day probably reflect variations in secretory mechanisms in the glands.

As suggested by Dawes ~'7~, the

changes in salivary composition are difficult to interpret since the variations in composition could be due to (a) changes in plasma composition with time of day, (b) changes in the secretory mechanisms of the gland or (c) differences in the mean flow rate or irregular,-,

~,(~

9

. . . . . . . .

, . . . . . . . . .

3.0

, . . . . . . . . .

2.0

, . . . . . . .

1.0

PPM

Fig. 4 High field region of the proton NMR spectra of human parotid saliva (a) and submandibular saliva (b) and (e) of patients suspected with sialoadenitis measured at 360MHz at room temperature. ChemicaI shifts are shown relative to that of TSP. 320 transients were collected. 10

ities in flow rate during collection of saliva. Nevertheless, if saliva is collected at the same time of day and at the same flow rate, the N M R spectrum should be useful for detecting change in the secretory mechanism or in metabolism in the salivary glands. Fig. 4 shows the high field region of proton N M R spectra of parotid (a) and sub-

mandibular

saliva

(b)

and

(c)

of

three

d i f f e r e n t p a t i e n t s s u s p e c t e d of s i a l o a d e n i t i s . O n s i a l o g r a p h y n o n e of t h e m w a s a n t i c i p a t e d to b e inflamed.

This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. First presented at the 29th annual meeting of Dental Radiology, Hokkaido, JAPAN September 1988.

However, the spectra were

s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e of h e a l t h y

References

subjects.

1 ) Nummi, P. Alanen, A., Nanto, V. and Kormano, M.: Effect of hemolysis and clotting on proton relaxation times of blood. Acta Radiol. Diagn. 27 : 225-230, 1986 2 ) Raftos, J. E., Chapman, B. E., Knchel, P. W., Lovric, V~ A. and Stewart, I. M.: Intra-and extra erythrocyte pH at 37~and during long term storage at 4": phosphorus31 NMR measurements and an electrochemical model of the system. Haematologia 19 :' 251-268, 1986 3 ) Martinez, D. and Silvidi, A. A.: A nuclear magnetic resonance study of sodium in the saliva of patients with cystjc fibrosis of the pancreas. Arch. Biochem. Biophys. 148 : 224-227, 1972 4 ) Rebouche, C. J., Pearson, G. A., Serfass, R. E., Roth, C. W. and Finley, J. W.: Evaluation of nuclear magnetic resonance spectroscopy for determination of deuterium abundance in body fluids: Application to measurement of total-body water in human infants. Am. J. Clin. Nutr. 45 : 373-380, 1987 5 ) Harada, H.,' Shimizu, H. and Maeiwa, M.: 1H NMR of human saliva. A n application of N M R ~pectroscopy in forensic science. 34 : 189-195, 1987 6 ) Dawes, C.: Circadian rhythms in human salivary flow rate and composition. J. Physiol. 220 : 529-545, 1972 7 ) Dawes, C. and Chebib, F. S.: The influence of previous stimulation and the day of the week on the concentrations of protein and the main electrolytes in human parotid saliva. Archs. Oral Biol. 17 : 1289-1301, 1972 8 ) Dawes, C. and Ong, B. Y.: Circadian rhythms in ~the concentrations of protein and the main electrolytes in human unstimulated parotid saliva. Archs. Oral Biol. 18 : 1233-1242, 1973

As indicated with the arrows

in

t h e figure s o m e of t h e s i g n a l s o b s e r v e d f o r a healthy person disappeared ones were observed.

and

additional

T h e s e s i g n a l s m a y be

characteristic to disease or could be attributed t o t h e

metabolites

medicinal therapy.

resulting

from

the

If t h e s e s i g n a l s a r e c h a r -

a c t e r i s t i c t o d i s e a s e , N M R w i l l b e useful as a supplemental

diagnosis

method

of

X-ray

examination. The main practical advantages technique

are

that

measurements

of this can

be

p e r f o r m e d w i t h o u t a n y p r e t r e a t m e n t and t h a t t h e y c a n b e m a d e in a s h o r t t i m e (less t h a n 30 min.) and w i t h s m a l l s a m p l e s (0.4 ml). paper

reports

only preliminary

a p p l i c a t i o n of N M R

This

studies

on

s p e c t r o s c o p y to s a l i v a

a n d f u r t h e r s t u d i e s a r e c l e a r l y r e q u i r e d on i n t e r p r e t a t i o n of s p e c t r a .

Nevertheless our

r e s u l t s s u g g e s t t h a t N M R s p e c t r a of p a r o t i d saliva

should

about

the

Further

provide

physiology

useful of the

information oral

s t u d i e s a r e in p r o g r e s s

cavity.

on h u m a n

parotid saviva from both healthy subjects and patients under various conditions.

Reprint requests to:

Atuko Y. NOSAKA, M.Sc.,Ph.D. Department of Oral Radiology, School of Dentistry, Tokushima University, 3-18-15, Kuramoto-cho, Tokushima 770, Japan 11