Lipid composition of human labial salivary gland secretions

Lipid composition of human labial salivary gland secretions

$3.00+0.00 0003-9969/83 1983 Arch ord Bid.Vol. 28.No. 8.pp.711-714, Printed Copyright in Great Britain. All rights reserved LIPID B. L. 0 1983 P...

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$3.00+0.00 0003-9969/83

1983 Arch ord Bid.Vol. 28.No. 8.pp.711-714, Printed

Copyright

in Great Britain. All rights reserved

LIPID

B. L.

0 1983 PergamonPress Ltd

COMPOSITION OF HUMAN LABIAL SALIVARY GLAND SECRETIONS SLOMIANY*, E. ZDEBSKA*, V. L. N. MURTY*,

A. SLOMIANY*,

K. PETROPOULOU? and

I. D. M.&NDELt *Gastroenterology Research Laboratory, Department of Medicine, New York Medical College, Research Center, Metropolitan Hospital, New York, NY 10029 and tDivision of Preventive Dentistry, School of Dental and Oral Surgery, Columbia University, New York, NY 10032, U.S.A.

Summary-Extraction of the dialysed and lyophilized labial saliva with chloroform/methanol yielded 423.8 If: 73.0 pg of lipids/ml of saliva, a level 4-5 times higher than in the major salivary glands. Of the total lipids, 32.4 per cent were represented by neutral lipids, 44.6 per cent by glycolipids and 23.0 per cent by phospholipids. Neutral lipids had a high content of free fatty acids (43.8 per cent), cholesteryl esters (26.9 per cent) and triglycerides (15.4 per cent). The glycolipids cclnsisted mainly of neutral (74.6 per cent) and sulphated (25.4 per cent) glyceroglucolipids, whereas phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and phosphatidylserine accounted for 54.7 per cent of the total phospholipids.

INTRODUCTION Saliva is a complex mixture of secretions from serous, mucous and mixed salivary glands located in the mouth and consists of proteins, glycoproteins, lipids and inorganic ions (Dirksen, 1963; Mandel, 1974, 1977; Prakobphol et d., 1982). The lipids in saliva are represented by neutr.31 lipids, glycolipids of glyceroglucolipid type and phospholipids (Mandel and Eisenstein, 1969; Rabinowitz and Shannon, 1975; Slomiany, Slomiany and Mandel, 1980, 1981; Slomiany et a!., 1982a, b). These data come mainly from the studies on the secretions of parotid and submandibular salivary glands, and only limited information exists on the lipids in the secretions of minor salivary glands (Hensten-Petterson, 1976). The secretions of the major salivary glands are either serous (parotid) or only partially mucous (submandibular) and none of the studies to date provide information about the lipids in the secretions of mucous glands. For this reason, we have chosen to investigate the content and composition of lipids in the predominantly mucous secretions of labial salivary glands.

MATERIAL AND

METHODS

Labial salivary gland secretions were obtained from 10 young adults over the age of 24 years with no clinical evidence of salivary gland pathology. The lower labial mucosa, exposed by everting the lip, was thoroughly dried with a cotton-roll and secretion from labial glands was stimulated with 2 per cent cirtic acid applied to the tongue. The droplets of secretion which appeared on the surface of the lip were collected by micropipette. Individual collections ([email protected] ml/subject) were divided into two equal 711

pools. Each pool was dialysed against distilled water, lyophilized and its protein content was determined (Lowry et al., 1951). Lipid analysis Dried samples were extracted twice, each time for 24 h, with chloroform-methanol (2: 1, v/v) and filtered through a grade-F sintered-glass funnel to retain insoluble protein residue (Slomiany et al., 1980). Fractionation of lipids contained in the extracts into neutral lipids, glycolipids and phospholipids was accomplished by column (0.5 x 5 cm) chromatography on silicic acid (Slomiany et al., 1980). Neutral lipids were separated into individual components by thinlayer chromatography, identified by comparison with chromatograms of authentic standards and quantitated (Slomiany et al., 1978, 1982a, b). The glycolipids were chromatographed on DEAE-Sephadex into neutral and acidic fractions, and were then separated into individual components by thin-layer chromatography (Slomiany et al., 1980). Following differentiation into glycosphingolipids and glyceroglucolipids by visualization with benzidine and orcinol reagents, the individual compounds were quantitated by gas-liquid chromatography (Slomiany et al., 1982a, b). The phospholipids were separated into individual compounds by two-dimensional thin-layer chromatography (Slomiany et ul., 1978, 1982a, b), identified by co-chromatography with appropriate standards and quantitated by the procedure of Lowry and Tinsley (1974). Gasliquid chromatography was performed with a Beckman GC-65 instrument equipped with glass columns (180 x 0.2 cm) packed with 3 per cent SE-30 on GasChrom-Q. For the analysis of fatty acid methyl esters, the temperature was programmed at 2”C/min from 170 to 290°C. The temperature programme for trimethylsilyl derivatives of glycerol and methyl glycosides was 10&2 lO”C/min.

712

B. L. Slomiany

Table

1. Content of protein and lipids in the labial salivary gland secretions pg/mf of secretions

Constituent Protein Total lipids* Neutral lipids? Glycolipids Phospholipids

1183.2 423.8 130.4 179.3 92.1

k & + k k

206.4 73.0 22.2 20.4 11.5

Each value represents means _tSD of duplicate analyses performed on two different pools. * Determined gravimetrically. t By summation of individual neutral lipid components. RESULTS

Extraction of dialysed and lyophilized labial saliva with chloroform-methanol yielded 423.8 + 73.0 ng of lipids per 1 ml of saliva. Following fractionation of the lipid extracts on silicic acid columns, 32.4 per cent of lipids were in the neutral lipid fraction, 44.6 per cent in the glycolipid fraction and 23.0 per cent in the phospholipid fraction (Table 1). Thin-layer chromatography of the neutral lipids revealed the presence of free fatty acids, mono-, diand triglycerides, cholesterol and cholesteryl esters. The content of individual neutral lipids per 1 ml of labial saliva is given in Table 2. Of the total neutral lipids, 43.8 per cent were represented by free fatty acids, 26.9 per cent by cholesteryl esters, 15.4 per cent by triglycerides, 11.6 per cent by cholesterol and 2 per cent by mono- and diglycerides. Thin-layer chromatographic examination of the glycolipids eluted from silicic acid column, visualized with benzidine and orcinol reagents, revealed several orcinol-positive bands, two of which also reacted with benzidine, indicating that glycolipids of labial saliva consist of glyceroglucolipids and glycosphingolipids. These two lipids constituted 0.551.3 per cent of the total glycolipids and were comprised of glucosylceramide and lactosylceramide. The glyceroglucolipid composition of labial saliva is given in Table 3. Sulphated glyceroglucolipids accounted for 25.4 per cent of the glyceroglucolipid fraction and were represented by tri- and tetraglucosyl compounds. The neutral glyceroglucolipids were represented mainly by hexa-

ef d.

Table 3. Percentage composition of glyceroglucolipids in the labial salivary gland secretions Glyceroglucolipid (No. of Glc residues)

Sulphated _ _ _

1 2 4 6 8 3 4

+ +

Constituent

pg/ml of secretions

Free fatty acids Mono- and diglycerides Triglycerides Cholesterol Cholesteryl esters

57.1 2.9 20.3 15.2 35.1

f 8.4 k 0.5 If: 2.4 k 3.3 f 4.2

Each value represents means +SD of duplicate analyses performed on two different pools of saliva.

5.2 1.0 6.8 48.1 13.5 20.6 4.8

Values represent the means of duplicate performed on the combined pools of saliva.

analyses

and octaglucosyl glyceroglucolipids. These two compounds constituted 61.6 per cent of the total glyceroglucolipids. The proportions of individual phospholipid classes present in labial saliva are shown in Table 4. The phospholipids constituted 23 per cent of labial saliva lipids and were comprised mainly of phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and phosphatidylserine. These four compounds accounted for 54.7 per cent of the labial saliva phospholipids. The fatty-acid composition of labial saliva lipids is given in Table 5. Free fatty acids and cholesteryl esters exhibited a high content of hexadecanoate, octadecanoate and docosenoate. The triglycerides contained large quantities (67.7 per cent) of hexadecanoate, octadecanoate and docosenoate. The glycolipids were rich in octadecanoate (30.1 per cent) and docosenoate (19 per cent), whereas octadecanoate (38.9 per cent) was the major fatty acid of the phospholipid fraction.

DISCUSSION The salivary glands in man are divided functionally into serous, mucous and mixed salivary glands. The

Table 4. Phospholipid composition gland secretions

Phospholipid Table 2. Composition of neutral lipids in the labial salivary gland secretions

Mol/lOO mol of glyceroglucolipids

Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Sphingomyelin Lysophosphatidylcholine Lysophosphatidylethanolamine Phosphatidic acid Phosphatidylglycerol Diphosphatidylglycerol Unidentified

of labial salivary

Percentage ot total lipid P 16.3 13.8 11.4 8.8 13.2 6.9 1.3 8.1 5.2 7.8 7.2

Values represent the means of duplicate performed on the combined pools of saliva.

analyses

Lipids of labial saliva Table 5. Fatty acid composition Fatty acid*

Free fatty acids

12:o 14:o 16:0 16:l 18:0 18:l 18 : Oa-OH 20:o 22:l Unidentified

6.5 0.2 20.9 6.7 18.7 17.5 0.5 10.0 14.1 4.9

Cholesteryl

of labial saliva lipids

Triglycerides esters Percentage of total

1.2 0.7 24.5 7.3 25.2 16.0 2.9 8.1 8.4 5.7

713

4.3 0.5 23.5 8.9 29.8 6.5 1.2 6.3 14.4 4.6

Glycolipids

2.1 0.3 16.8 5.4 30.1 11.0 0.3 10.2 19.0 4.8

* The number before the colon denotes the number of carbon atoms, the number number of double bonds, and cc-OH refers to 2 hydroxy fatty acids.

sublingual and minor salivary glands contain mostly mucous acinar cells. The parotid glands are purely serous, whereas the acinar cells of submandibular glands are 75 per cent serous and 25 per cent mucous (Mandel, 1974, 1977). About 90 per cent of the secreted salivary fluids z.re derived from the parotid and submandibular glandr,, 5 per cent from the sublingual glands and 5 per cent from the minor salivary glands (Mandel and Wotman, 1976). The fluids secreted by the serous glands also differ considerably from those of mucous glands with respect to their composition. The secretions of serous glands contribute most of the salivary amylase and proline-rich cationic glycoproteins in whole saliva, while the mixed and mucous glands are the major source of the high-molecularweight mucus glycoproteins which are responsible for the viscoelastic propc:rties of saliva (Levine, Ellison and Bahl, 1973; Mandel, 1974, 1977; Prakobphol et al., 1982). The minor salivary glands including labial glands, although contributing only about 5 per cent to the volume of whole saliva, are the predominant source of mucus glycoproteins which are responsible for the blood-group activity in saliva (Milne and Dawes, 1973). However, considerably less is known about the lipids in the secretions of minor salivary glands. Although lipid inclus#ions in labial salivary glands were observed electron-microscopically (Tandler et al., 1969; Doggett, Bentinck and Harrison, 1971) and the presence of a polar lipid with chromatographic migration of phosphatidylinositol was reported (Hensten-Pettersen, 1976), no quantitative analysis of lipids in labial saliva was ever attempted. Our findings show l.hat the secretion of labial salivary glands contains 423.8 pg of lipids/ml of saliva. These lipids, like thost: of parotid and submandibular saliva, are represented by the neutral lipids, phospholipids and glycolipids. The neutral lipids constitute 32.4 per cent of the labial saliva lipids and are composed of free fatty acids (mainly hexadecanoate, octadecanoate and act adecenoate), cholesteryl esters, cholesterol and mono, di- and triglycerides. Glycolipids constitute 44.6 per cent of labial saliva lipids and in addition to glyc’eroglucolipids also contain 0.51.3 per cent of simple glycosphingolipids. Phospho-

Phospholipids

1.8 0.2 23.7 2.7 38.9 19.8 1.0 2.4 8.3 1.2 following

the colon is the

lipids represent 23.0 per cent of labial saliva lipids and are characterized by a high content of phosphatidylethanolamine, phosphatidylcholine sphingomyelin and phosphatidylserine. Comparison of the data on labial salivary lipids with those found in parotid and submandibular saliva (Slomiany et al., 1982a, b) indicates that labial saliva contains 4-5 times more lipids/ml of saliva, and exhibits a higher percentage of phospholipids and glycolipids. These differences may be a consequence of different processes by which serous and mucous cells liberate their products, or they may reflect the differences in the interaction between the lipids of glandular membrane and the proteins and glycoproteins of secretions. A high content of phospholipids and the presence of glycosphingolipids in labial saliva support the findings of Tandler and Poulsen (1976) that mucous cells liberate their products by a partly apocrine process during which part of the cell membrane is shed and excreted. Hence, the secretions of these cells would exhibit a high content of phospholipids and contain the glycosphingolipids, which are characteristic of cell membrane. In the light of our studies which showed that lipids in submandibular saliva exhibit a high affinity for mucus glycoproteins (Slomiany et nl., 1983), it is also possible that the glycoproteins elaborated by mucous cells of labial glands interact with the membrane lipids and that these lipid-enriched mucosubstances are secreted into the saliva. The high content of lipids in the secretions of labial salivary glands may be of importance in the protection of labial mucous membranes in the mouth, although direct evidence of this is not available. Studies on mucous secretions of gastrointestinal and respiratory tracts indicate that lipids contribute to physicochemical and functional properties of these secretions (Forstner, 1978; Martin, Marriot and Kellaway, 1978). The lipids in labial saliva may contribute importantly to rheological properties of mixed saliva. Acknowledgement-This work was supported by USPHS Grants DE No. 05666-02 and DE No. 01554-21 from the National Institute of Health.

of Dental

Research,

National

Institutes

714

B. L. Slomi lany et al. REFERENCES

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