Archs oral Biol.
Vol. 36, NIL 9, pp. 697-701, 1991
0003~9969/91 53.00+ 0.00 copyright 0 1991Pcr&IamonPress plc
Rind in Chat Britain. All rights reserved
SHORT COMMUNICATION PRELIMINARY NUCLEAR MAGNETIC RESONANCE STUDIES ON HUMAN SALIVA ATSUKO YAMADA-N~~AKA,’ SHIGERUFUKUTOMI,’ SHLJSABIJRO UEMURA,’ TATSUO HASHIDA,* MASAMI FUJISHITA,*YUJI KOBAYASHI’and YOSHIMASAKY-KU’
‘Department of Oral Radiology, School of Dentistry, Tokushima University, Tokushima 770, ZDepartment of Oral and Maxillofacial Radiology and 31nstitute for Protein Research, Osaka University, Suita, Osaka 565, Japan (Accepted 12 March 1991) Summary-Of gustatory-stimulated human whole, parotid, submandibular and sublingual saliva only parotid saliva, a serous rather than mucous secretion, presented a relatively well-resolved proton NMR spectrum with satisfactory signal-to-noise ratio in a short time (30min). The proton signal intensities showed significant circadian rhythms related to the circadian rhythms of protein concentrations in saliva. Age- and sex-associated differences in spectra were not observed for healthy saliva. On the other hand, marked differences in the spectra were observed for patients with suspected sialoadenitis. Key words: proton NMR, human parotid saliva, human submandibular saliva, sialoadenitis.
Body fluids such as blood, urine, gastric juice, bile and pancreatic juice have been used extensively in diagnostic tests. NMR, which is recognized as a powerful tool for biochemical analysis, has been applied to blood (Nicholson and Wilson, 1989; Bell, Brown and Sadler, 1989), but the use of highresolution NMR spectroscopy for diagnostic tests is complicated because several pretreatments of blood are required. Saliva contains diffusion components that are basically similar to those of blood. However, as saliva collected from the mouth (whole saliva) is a mixture of secretisons and contains leucocytes, desquamated epithelial cells and oral bacteria, few NMR assays of it have been attempted (Martinez and Silvidi, 1972; Rebouche et al., 1987; Harada, Shimizu and Maeiwa, 1987; Coudert et al., 1989). If saliva is collected a:septically from the ducts of the parotid, submandibular or sublingual gland the above objections are overcome. We have now attempted to measure the NMR spectra of saliva and assess its diagnostic possibilities. Acid-stimulated h.uman parotid saliva (from subjects aged 26-42 yr) was collected through a Teflon cannula inserted into the orifice of Stensen’s duct. Submandibular and sublingual salivas were collected together through the orifice of Wharton’s duct as the two are difficult to collect separately. As well as cannulation a special collection device developed by Kuboki et al. (11982) was also used. There was no difference in spectra associated with the different methods of collection. About 2ml of saliva were collected during lo-30 min. There was no significant difference between the spectra of the first and second millilitre. Samples from 24 different subjects Abbreviation: NMR, nuclear magnetic resonance.
[8 healthy women, 8 healthy men, and 8 patients (see below)] were tested. The collected saliva was stored at 5°C for 2 days before measurement. The measurements were carried out in the Smm O.D. NMR sample tube with a Bruker WM-360 FT spectrometer operating in the Fourier transform mode at 360 MHz. As a spectrometer ‘lock’, 0.1 ml of D,O (99.5%) was added to 0.4ml of a sample. The pH of saliva, measured just after collection, was 6.9 f 0.6. All the NMR measurements were at room temperature. 3-(trimethylsilyl) propionate was used as an external standard for chemical shift and signal intensity. The HDO signal was irradiated for 3 s before signal acquisition to suppress the water peak. Unless otherwise stated, 320 transients were collected for about 30 min. Homonuclear-correlated (COSY) and homonuclear Hartmann-Hahn (HOHAHA) spectra were measured at 500 MHz on a JEOL GX-500 spectrometer with the standard pulse sequences. The HOHAHA spectra were measured with a mixing time of 66ms. Saliva was lyophilized and dissolved in D,O before two-dimensional NMR measurements to eliminate the effect of the water signal. Residual HDO was suppressed by weak irradiation during the pulse delay. Viscosity measurements were performed in a TOKYO KEIKI cone/plate digital viscometer, model Visconic ELD-R, at room temperature. All measurements were repeated three times for reproducibility. Proton NMR spectra of healthy human saliva
These spectra of whole saliva and the mixed submandibular/sublingual saliva were broad and not well resolved, reflecting the higher viscosity of mucous components. Parotid saliva presented a well697
ATSUKO YAMADA-NOSAKA et al.
698
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Fig. 1. The high-field region of proton NMR spectra of human parotid saliva measured at 360 MHz at room temperature, collected at (a) 10: 30, (b) 13: 30, (c) 15: 30 and (d) 19: 30.
resolved spectrum with a satisfactory signal-to-noise ratio within 30min (Fig. 1). Only the high-field regions are presented here as they were well resolved and gave a good signal-to-noise ratio, although the low-field, aromatic regions could provide also important information. With larger numbers of accumulations (about 8 h), whole and mixed salivas presented similar but broader spectral features to those of parotid saliva, indicating the same visible components as those of the parotid saliva but at lower concentration.
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This suggests that the proton NMR detects mainly serous components of saliva. The proton NMR spectra were not different in relation to age or sex, or flow rate. The spectra measured 1 h, 9 h and 2 days after collection were similar except for a peak at 1.2 parts/lo6 which disappeared in the spectrum measured 2 days after collection. Figure 1 shows the high-field region of the proton NMR spectra of parotid salivas collected at different times of the day. The intensities of most of the signals
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Fig. 2. The high-field region of proton NMR spectra of human parotid saliva measured at 360 MHz at room temperature with inversion recovery mode at an interval time of 0.4 s.
Preliminary NMR studies on human saliva
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Vol. 36, NIL 9, pp. 697-701, 1991
0003~9969/91 53.00+ 0.00 copyright 0 1991Pcr&IamonPress plc
Rind in Chat Britain. All rights reserved
SHORT COMMUNICATION PRELIMINARY NUCLEAR MAGNETIC RESONANCE STUDIES ON HUMAN SALIVA ATSUKO YAMADA-N~~AKA,’ SHIGERUFUKUTOMI,’ SHLJSABIJRO UEMURA,’ TATSUO HASHIDA,* MASAMI FUJISHITA,*YUJI KOBAYASHI’and YOSHIMASAKY-KU’
‘Department of Oral Radiology, School of Dentistry, Tokushima University, Tokushima 770, ZDepartment of Oral and Maxillofacial Radiology and 31nstitute for Protein Research, Osaka University, Suita, Osaka 565, Japan (Accepted 12 March 1991) Summary-Of gustatory-stimulated human whole, parotid, submandibular and sublingual saliva only parotid saliva, a serous rather than mucous secretion, presented a relatively well-resolved proton NMR spectrum with satisfactory signal-to-noise ratio in a short time (30min). The proton signal intensities showed significant circadian rhythms related to the circadian rhythms of protein concentrations in saliva. Age- and sex-associated differences in spectra were not observed for healthy saliva. On the other hand, marked differences in the spectra were observed for patients with suspected sialoadenitis. Key words: proton NMR, human parotid saliva, human submandibular saliva, sialoadenitis.
Body fluids such as blood, urine, gastric juice, bile and pancreatic juice have been used extensively in diagnostic tests. NMR, which is recognized as a powerful tool for biochemical analysis, has been applied to blood (Nicholson and Wilson, 1989; Bell, Brown and Sadler, 1989), but the use of highresolution NMR spectroscopy for diagnostic tests is complicated because several pretreatments of blood are required. Saliva contains diffusion components that are basically similar to those of blood. However, as saliva collected from the mouth (whole saliva) is a mixture of secretisons and contains leucocytes, desquamated epithelial cells and oral bacteria, few NMR assays of it have been attempted (Martinez and Silvidi, 1972; Rebouche et al., 1987; Harada, Shimizu and Maeiwa, 1987; Coudert et al., 1989). If saliva is collected a:septically from the ducts of the parotid, submandibular or sublingual gland the above objections are overcome. We have now attempted to measure the NMR spectra of saliva and assess its diagnostic possibilities. Acid-stimulated h.uman parotid saliva (from subjects aged 26-42 yr) was collected through a Teflon cannula inserted into the orifice of Stensen’s duct. Submandibular and sublingual salivas were collected together through the orifice of Wharton’s duct as the two are difficult to collect separately. As well as cannulation a special collection device developed by Kuboki et al. (11982) was also used. There was no difference in spectra associated with the different methods of collection. About 2ml of saliva were collected during lo-30 min. There was no significant difference between the spectra of the first and second millilitre. Samples from 24 different subjects Abbreviation: NMR, nuclear magnetic resonance.
[8 healthy women, 8 healthy men, and 8 patients (see below)] were tested. The collected saliva was stored at 5°C for 2 days before measurement. The measurements were carried out in the Smm O.D. NMR sample tube with a Bruker WM-360 FT spectrometer operating in the Fourier transform mode at 360 MHz. As a spectrometer ‘lock’, 0.1 ml of D,O (99.5%) was added to 0.4ml of a sample. The pH of saliva, measured just after collection, was 6.9 f 0.6. All the NMR measurements were at room temperature. 3-(trimethylsilyl) propionate was used as an external standard for chemical shift and signal intensity. The HDO signal was irradiated for 3 s before signal acquisition to suppress the water peak. Unless otherwise stated, 320 transients were collected for about 30 min. Homonuclear-correlated (COSY) and homonuclear Hartmann-Hahn (HOHAHA) spectra were measured at 500 MHz on a JEOL GX-500 spectrometer with the standard pulse sequences. The HOHAHA spectra were measured with a mixing time of 66ms. Saliva was lyophilized and dissolved in D,O before two-dimensional NMR measurements to eliminate the effect of the water signal. Residual HDO was suppressed by weak irradiation during the pulse delay. Viscosity measurements were performed in a TOKYO KEIKI cone/plate digital viscometer, model Visconic ELD-R, at room temperature. All measurements were repeated three times for reproducibility. Proton NMR spectra of healthy human saliva
These spectra of whole saliva and the mixed submandibular/sublingual saliva were broad and not well resolved, reflecting the higher viscosity of mucous components. Parotid saliva presented a well697
ATSUKO YAMADA-NOSAKA et al.
698
(a)
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parts
0
1
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/IO6
Fig. 1. The high-field region of proton NMR spectra of human parotid saliva measured at 360 MHz at room temperature, collected at (a) 10: 30, (b) 13: 30, (c) 15: 30 and (d) 19: 30.
resolved spectrum with a satisfactory signal-to-noise ratio within 30min (Fig. 1). Only the high-field regions are presented here as they were well resolved and gave a good signal-to-noise ratio, although the low-field, aromatic regions could provide also important information. With larger numbers of accumulations (about 8 h), whole and mixed salivas presented similar but broader spectral features to those of parotid saliva, indicating the same visible components as those of the parotid saliva but at lower concentration.
I
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I
I 3
I
This suggests that the proton NMR detects mainly serous components of saliva. The proton NMR spectra were not different in relation to age or sex, or flow rate. The spectra measured 1 h, 9 h and 2 days after collection were similar except for a peak at 1.2 parts/lo6 which disappeared in the spectrum measured 2 days after collection. Figure 1 shows the high-field region of the proton NMR spectra of parotid salivas collected at different times of the day. The intensities of most of the signals
I 2
Parts
I
I 1
I
I 0
/ IO’
Fig. 2. The high-field region of proton NMR spectra of human parotid saliva measured at 360 MHz at room temperature with inversion recovery mode at an interval time of 0.4 s.
Preliminary NMR studies on human saliva
S -0 ?
r-u-.
