Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples

Forensic Science International 129 (2002) 58±63

Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples W. Kim, Y.K. Kim, S.C. Chung, S.W. Lee, H.S. Kho* Department of Oral Medicine & Oral Diagnosis, College of Dentistry, Seoul National University, 28-22 Yunkeun-Dong, Chongro-Ku, Seoul 110-744, South Korea Received 27 February 2002; received in revised form 25 June 2002; accepted 27 June 2002

Abstract The purpose of the present study was to identify salivary molecules carrying the ABH blood group antigens in Koreans and to investigate the changes in these antigens according to processing and storage of saliva samples. Secretor or non-secretor phenotypes and salivary components carrying the ABH antigens were identi®ed in 90 subjects, 30 subjects in each ABO blood group, by SDS-PAGE and immunoblotting. Saliva samples were then obtained from 12 secretorsÐtwo males and two females in each ABO blood group and aliquots of both fresh saliva samples and their supernatants after centrifugation were stored at room temperature, 4, 20 and 70 8C. The same experiments were performed after 1, 3 and 6 months to investigate changes in the blood group antigens. In all 68 secretors, high-molecular-weight salivary mucin (MG1) was found to be the primary carrier of the ABH antigens. A salivary component of approximately 80 kDa also carried H antigen in seven saliva samples of 22 blood type O secretors. The blood group antigens were better detected in centrifuged samples. In saliva samples preserved at room temperature and 4 8C, the blood group antigens were either not detected or detected as degraded molecules. No change was found in the blood group antigens in saliva samples preserved at 20 and 70 8C for 6 months. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Saliva; Blood group antigens; Mucin

1. Introduction Saliva plays important roles in the oral cavity, including mechanical ¯ushing, lubrication, protection of oral tissues, modulation of oral micro¯ora, neutralization of deleterious materials, regulation of calcium/phosphate equilibrium, digestion and taste perception. These functions can be attributed, in part, to various salivary proteins and glycoproteins [1]. In the case of salivary glycoproteins, their biological and functional characteristics are mainly determined by their carbohydrate moieties, which play important roles in the interaction between salivary glycoproteins and oral microorganisms [2]. In addition, the carbohydrate moieties of salivary glycoproteins can carry the ABH blood group antigens, which are expressed in the salivary glands * Corresponding author. Tel.: ‡82-2-760-2611 (O); fax: ‡82-2-744-9135. E-mail address: [email protected] (H.S. Kho).

and secreted in the saliva [3±5]. Thus, the expression of the blood group antigens in saliva might alter the speci®c interactions between microorganisms and their salivary glycoprotein receptors, and this might intervene in the development and prevention of oral infectious diseases [4,6,7]. Some individuals, so-called secretors, possess the ability to secrete blood group substances in secretions, such as saliva, tears and gastric secretions. However, a percentage of people, the so-called non-secretors, lack this ability and secrete substances devoid of these blood group antigens. The percentage ratio of secretors to non-secretors has been reported to be approximately 80 to 20% [5]. It is generally accepted that the glycosylation and expression pattern of the ABH blood group antigens are genetically diverse and determined by the ABH, Se and Le genes, and that the genetic frequency is different among ethnic groups [8]. It has been also suggested that salivary mucins, highly glycosylated glycoproteins, which play important roles in

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W. Kim et al. / Forensic Science International 129 (2002) 58±63

lubrication, tissue protection and modulation of micro¯ora in the oral cavity [9±11], are carriers of the blood group antigens [4]. In particular, high-molecular-weight salivary mucin (MG1) was found to be the principal carrier of the oligosaccharide moieties that constitute the ABH, Lea and Leb blood group antigens and low-molecular-weight salivary mucin (MG2) also carried these blood group antigens [4]. Considering the limited number of subjects in the previous study [4] and ethnic variation in the expression of the blood group antigens, it is worthwhile to investigate salivary components, which serve to express and carry the ABH blood group antigens from larger number of subjects of different ethnic group. It was demonstrated that the ABH and Lewis antigens could be determined from urine samples stored over 18 months [12]. The ABH, Lewis and other blood group antigens of red blood cells could survive for 6 months to 1 year after the preparation of ghosts or freeze-dried cells [13]. However, some changes might occur in terms of the detection of the ABH blood group antigens in saliva samples because of the progressive degradation of salivary components carrying the blood group antigens by enzymes that originate from bacteria and serum. The purpose of the present study was to identify the salivary components which carry the ABH blood group antigens in the saliva of Koreans, and to investigate changes in the detection of the blood group antigens in saliva according to the preparation method used, the storage temperature and the storage period of saliva samples. 2. Materials and methods 2.1. Subjects Ninety students of the College of Dentistry, Seoul National University whose ABO blood types were identi®ed were included in the study. The number of subjects in each ABO blood type were 30 (male: 15, female: 15), respectively. Their mean age ranged from 22 to 26 years (mean age 23:7  0:8 years). 2.2. Collection of unstimulated whole saliva Unstimulated whole saliva was collected by a standard, reproducible method [14]. Brie¯y, samples from the subjects were collected between 9.00 and 11.00 h to minimize diurnal variability in salivary composition. All subjects were requested to refrain from eating, drinking and brushing for at least 2 h prior to sample collection. Unstimulated whole saliva was collected for about 5 min by the spitting method (i.e. after swallowing, saliva is collected with closed lips, and then all saliva is expectorated into a vessel one or two times per min) [15]. Each saliva sample was collected into a chilled sterile centrifuge tube and centrifuged at 3500  g for 5 min to remove cellular debris.

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Saliva samples from the supernatants were then used immediately for the experiment. 2.3. Electrophoresis and immunoblotting Saliva samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with the use of an electrophoresis unit (SE 280 Tall Mighty Small; Hoefer Scienti®c Instruments, San Francisco, CA). Western blotting was performed according to previously reported method [16,17]. After transferring salivary proteins to Immobilon-P membranes (polyvinylidene di¯uoride [PVDF] membranes; Millipore Corp., Bedford, MA) using a Fisher Biotech Semi-Dry Blotting Unit 2020 (Fisher Scienti®c, Pittsburgh, PA) at 300 mA for 45±60 min, unbound sites on the membrane were blocked with Trisbuffered saline (TBS: 10 mM of Tris±HCl, 154 mM of NaCl, pH 7.5) containing 3% bovine serum albumin (BSA) for more than 1 h. Membranes were then incubated with a 1:100 dilution of monoclonal mouse anti-human blood group antigens A, B and H (DAKO Corp., Carpinteria, CA) as primary antibodies for 3 h at room temperature. After being washed three times for 5 min each with TBS containing 0.01% BSA, the blots were incubated with a 1:1000 dilution of horseradish peroxidase conjugate rabbit anti-mouse IgG (DAKO Corp.) as a secondary antibody for 2 h. The same washing cycle with TBS containing 0.01% BSA was then repeated, and the blots were visualized with the solution of 6 mg of DAB (diaminobenzidine tetrahydrochloride, DAKO Corp.) in 10 ml of 50 mM Tris±HCl buffer (pH 7.6) by adding 100 ml of 3% H2O2. All test samples were assayed at least twice in the same manner. The saliva samples of subjects, identi®ed as secretors, were collected immediately before each experiment and used as positive controls. To exclude the possibility of cross-reactivity between the ABH blood group antigens, the same experiments were performed using antibodies to other blood groups as primary antibodies. For negative controls, the same experiments were performed without applying primary antibodies. For the detection of high-molecular-weight salivary mucin (MG1) in saliva samples, a 1:1000 dilution of rabbit anti-MG1 polyclonal sera (kindly donated by Dr. Michael J. Levine, Department of Oral Biology, State University of New York at Buffalo) was used as a primary antibody and a 1:2500 dilution of horseradish peroxidase conjugate goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA) was used a secondary antibody. 2.4. Effect of the preparation method, storage temperature and storage period of the saliva samples Four subjects (two males and two females) in each A, B and O blood group identi®ed as secretors in the previous experiment were included in this experiment. Unstimulated whole saliva samples were collected from the four secretor

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W. Kim et al. / Forensic Science International 129 (2002) 58±63

subjects in each blood group over a period of 15 min. Half of sample (about 3 ml) was centrifuged at 3500  g for 5 min to remove cellular debris and the other half was not. Aliquots were made from both kinds of samples and stored at room temperature (about 25 8C), 4, 20 and 70 8C, and after periods of 1, 3 and 6 months, the same experiment was performed at least twice to determine whether any changes in the detection of ABH blood group antigens in the saliva samples had occurred. 3. Results 3.1. Percentage of secretor versus non-secretor The percentage of secretor versus non-secretor phenotype in a total of 90 subjects was 75.6% versus 24.4%, and the percentages in each A, B and O individuals were 90.0% versus 10.0%, 69.4% versus 30.6% and 73.3% versus 26.7%, respectively.

Fig. 1. SDS-PAGE (10%)/Western blotting of saliva samples. Immunoblotting of electrophoretically separated saliva samples with monoclonal mouse antibodies to A, B and H blood group antigens as primary antibodies (Lanes 2±8). A salivary component carrying ABH blood group antigens was identi®ed as highmolecular-weight salivary mucin (MG1) by applying rabbit antiMG1 polyclonal antibody as a primary antibody (Lane 1). Lane 1, human unstimulated whole saliva (UWS); Lane 2, UWS of blood group A secretor; Lane 3, UWS of blood group A non-secretor; Lane 4, UWS of blood group B secretor; Lane 5, UWS of blood group B non-secretor; Lane 6, UWS of blood group O secretor; Lane 7, UWS of blood group O secretor (A molecule of approximately 80 kDa also carried the H antigen.); Lane 8, UWS of blood group O non-secretor.

3.2. Salivary component carrying the ABH blood group antigens When saliva samples transferred to the membrane were exposed to monoclonal antibodies to the ABH blood group antigens, the molecule located in the stacking gel showed positive signal in all ABO blood groups (Fig. 1). The molecule was identi®ed as MG1 by immunoblotting using rabbit anti-MG1 polyclonal antibody as a primary antibody (Fig. 1, Lane 1). In all 68 secretors among the 90 subjects, the salivary component carrying the ABH blood group antigens was identi®ed as MG1. In seven (31.8%) saliva samples of 22 blood type O secretor subjects, a molecule of approximately 80 kDa also carried the H antigen (Fig. 1, Lane 7). 3.3. Changes in the detection of the ABH blood group antigens according to the preparation method, storage temperature and storage period of the saliva samples In the case of saliva samples preserved at room temperature, the ABH blood group antigens were better detected in the centrifuged samples than in the non-centrifuged ones (Table 1). After storage for 6 months at 4 8C, H antigen was

Fig. 2. SDS-PAGE (10%)/Western blotting of saliva samples from one blood group A secretor preserved at room temperature for 1, 3 and 6 months. Blood group A antigen was not detected in either the centrifuged or non-centrifuged samples after 3 and 6 months. Lane 1, positive control (fresh unstimulated whole saliva from blood group A secretor); Lane 2, centrifuged sample after 1 month; Lane 3, non-centrifuged sample after 1 month; Lane 4, centrifuged sample after 3 months; Lane 5, non-centrifuged sample after 3 months; Lane 6, centrifuged sample after 6 months; Lane 7, noncentrifuged sample after 6 months.

W. Kim et al. / Forensic Science International 129 (2002) 58±63

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Table 1 Detection of the ABH blood group antigens in saliva samples preserved at room temperature for 1, 3 and 6 months Blood type

Subject (n ˆ 12)

1 month

3 months

6 months

Centrifuged sample

Non-centrifuged sample

Centrifuged sample

Non-centrifuged sample

Centrifuged sample

Non-centrifuged sample

A

Male 1 Male 2 Female 1 Female 2

‡ (‡) ‡ (‡)

‡ (‡) ‡ (‡)

‡ (‡) (‡)

(‡) (‡) (‡)

(‡) (‡)

(‡)

B

Male 1 Male 2 Female 1 Female 2

‡

‡

(‡)

(‡)

(‡)

(‡)

‡

‡

(‡)

(‡)

(‡)

(‡)

Male 1 Male 2 Female 1 Female 2

‡ (‡) (‡) (‡)

‡

‡ (‡)

‡

(‡)

(‡)

O

(‡)

‡: positive response; (‡): positive as degraded state;

: negative response.

Table 2 Detection of the ABH blood group antigens in saliva samples preserved at 4 8C for 1, 3 and 6 months Blood type

Subject (n ˆ 12)

1 month

3 months

6 months

Centrifuged sample

Non-centrifuged sample

Centrifuged sample

Non-centrifuged sample

Centrifuged sample

Non-centrifuged sample

A

Male 1 Male 2 Female 1 Female 2

‡ ‡ ‡ ‡

‡ ‡ ‡ ‡

‡ (‡) (‡) (‡)

‡ (‡) (‡) (‡)

‡ (‡) (‡) (‡)

‡ (‡) (‡) (‡)

B

Male 1 Male 2 Female 1 Female 2

‡ (‡)

‡ (‡)

‡

‡

(‡)

(‡)

‡

‡

(‡)

(‡)

(‡)

(‡)

O

Male 1 Male 2 Female 1 Female 2

‡ ‡ ‡ ‡

‡ ‡ (‡) ‡

‡ ‡ (‡) ‡

‡ ‡ (‡) (‡)

‡ (‡) (‡) (‡)

(‡) (‡) (‡)

‡: positive response; (‡): positive as degraded state;

: negative response.

detected only in the case of centrifuged samples in one of four samples (Table 2). After 1 and 3 months, the H antigen in one of four samples was detected as degraded molecules in non-centrifuged samples and as an intact molecule in centrifuged ones (Table 2). The centrifugation of saliva samples did not affect the detection of the ABH blood group antigens in saliva preserved at 20 and 70 8C. The ABH blood group antigens were better detected in the saliva samples preserved at lower temperature. In the case of saliva samples preserved at room temperature and 4 8C, the blood group antigens of some samples were either not detected or detected as degraded molecules as the storage

period was extended (Tables 1 and 2, Figs. 2 and 3). This tendency was more marked in the saliva samples preserved at room temperature than 4 8C (Tables 1 and 2). No change was found in the detection of the ABH blood group antigens in saliva samples preserved at 20 and 70 8C for at least 6 months (Fig. 4). 4. Discussion In the present study, MG1 was identi®ed as only carrier of blood group antigens in the saliva of blood group A and B

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Fig. 3. SDS-PAGE (10%)/Western blotting of saliva samples from one blood group B secretor preserved at 4 8C for 1, 3 and 6 months. Blood group B antigen was detected as degraded molecules both in the centrifuged and non-centrifuged samples. Lane 1, positive control (fresh unstimulated whole saliva from blood group B secretor); Lane 2, centrifuged sample after 1 month; Lane 3, noncentrifuged sample after 1 month; Lane 4, centrifuged sample after 3 months; Lane 5, non-centrifuged sample after 3 months; Lane 6, centrifuged sample after 6 months; Lane 7, non-centrifuged sample after 6 months.

secretors. In blood group O secretors, a molecule of approximately 80 kDa as well as MG1 carried H antigen in some samples. These results agree in part with a previous report by Prakobphol et al. [4] who reported that MG1 is the primary carrier of the oligosaccharide moieties that constitute the ABH, Lea and Leb blood group substances. They also found that MG2 expresses these antigens in some individuals. However, we could not observe this phenomenon in the Korean subjects included in the study. This difference could be attributed to ethnic variations in blood group antigen expression in salivary secretions. Considering the previous report on the glycoproteins in human parotid saliva [18], the approximately 80 kDa band showing H activity in seven subjects is related to proline-rich glycoprotein (PRG), one of the major glycoproteins of parotid saliva. In the present study, the results showed that the ABH blood group antigens were better detected in the centrifuged samples than in the non-centrifuged ones, especially in the case of saliva samples preserved at room temperature. These results were expected because the centrifugation of samples removes most of the cellular debris and bacteria, which produce the enzymes responsible for the progressive degradation of salivary components carrying the blood group antigens. The ABH blood group antigens were also better

Fig. 4. SDS-PAGE (10%)/Western blotting of saliva samples from one blood group O secretor preserved at 20 8C for 1, 3 and 6 months. Blood group H antigen was detected as an intact molecule both in the centrifuged and non-centrifuged samples. Lane 1, positive control (fresh unstimulated whole saliva from blood group O secretor); Lane 2, centrifuged sample after 1 month; Lane 3, non-centrifuged sample after 1 month; Lane 4, centrifuged sample after 3 months; Lane 5, non-centrifuged sample after 3 months; Lane 6, centrifuged sample after 6 months; Lane 7, noncentrifuged sample after 6 months.

detected when the saliva samples were preserved at lower temperature. In particular, the blood group antigens were detected in all saliva samples preserved at 20 and 70 8C for at least 6 months. However, enzymes originated from serum and bacteria may function in saliva samples preserved at room temperature or at 4 8C and the detection of the blood group antigens in such samples depended on individual sample as the storage period was extended. Thus, a positive signal was detected at lower molecular weight level than intact MG1, which means the degradation of MG1, the carrier of the ABH blood group antigens. As expected, this phenomenon was more marked in saliva samples preserved at room temperature than at 4 8C. 5. Conclusions The performed investigations indicated that high-molecular-weight salivary mucin is the primary carrier of ABH blood group antigens in Koreans. The ABH blood group antigens were better detected in centrifuged samples stored at lower temperature. These results also indicated a distinct difference in the structure and function of high-molecularweight salivary mucin between secretors and non-secretors.

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This ®nding could be used for further researches on the relationship between the blood group antigens and oral diseases as well as on its forensic applications. Acknowledgements This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea, HMP-00-CH-10-0009. References [1] I.D. Mandel, The functions of saliva, J. Dent. Res. 66 (1987) 623±633. [2] M.J. Levine, M.S. Reddy, L.A. Tabak, R.E. Loomis, E.J. Bergey, P.C. Jones, R.E. Cohen, M.W. Stinson, I. Al-Hashimi, Structural aspects of salivary glycoproteins, J. Dent. Res. 66 (1987) 436±441. [3] K. Hamper, J. Caselitz, G. Seifert, R. Seitz, A. Poschmann, The occurrence of blood group substances (A, B, H, Le-a, Le-b) in salivary glands and salivary gland tumors. An immunohistochemical investigation, J. Oral Pathol. 15 (1986) 334±338. [4] A. Prakobphol, H. Lef¯er, S.J. Fisher, The high-molecularweight human mucin is the primary salivary carrier of ABH, Lea, and Leb blood group antigens, Crit. Rev. Oral Biol. Med. 4 (1993) 325±333. [5] P. Greenwell, Blood group antigens: molecules seeking a function? Glycoconj. J. 14 (1997) 159±173. [6] A.P. Burford-Mason, J.C.P. Weber, J.M.T. Willoughby, Oral carriage of Candida albicans, ABO blood group and secretor status in healthy subjects, J. Med. Vet. Mycol. 26 (1988) 49±56. [7] P.-J. Lamey, A.M.G. Darwazeh, J. Muirhead, J.S. Rennie, L.P. Samaranayake, T.W. MacFarlane, Chronic hyperplastic candidosis and secretor status, J. Oral Pathol. Med. 20 (1991) 64±67.

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