Agents Actions, 35 (1992)
0065-4299/92/020029-05 $1.50+0.20/0 9 1992 Birkh/iuser Verlag, Basel
The influence o f age, sex and race on salivary kallikrein levels in human mixed saliva Joyce W. Jenzano 1,2, Susan L. Hogan 1, and R.L. Lundblad 1,a,4 Dental Research Center ~and Departments of Dental Ecology ~ and Periodolatics 3, School of Dentistry and Departments of Pathology and Biochemistry 4, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7455 (USA)
Abstract
Variation in the level of salivary kallikrein in human saliva has been reported as a function of systemic conditions such as reduced salt intake and during the menstrual cycle. Higher levels of salivary kallikrein have been observed in subjects with tumors distant from the oral cavity when compared to control subjects. These studies have not evaluated factors, such as age, which might influence the concentration of glandular kallikrein in saliva. The purpose of the present study was to determine the variation of salivary kallikrein concentration as a function of age. Differences attributable to sex or race were also evaluated. Mixed saliva was collected from 114 subjects, ages 5-91, by paraffin stimulation. Samples were centrifuged and stored at - 20 ~ for subsequent analysis. Glandular kallikrein activity was assayed using D-ValylLeucylArginine-p-nitroanilide as the substrate. In a linear regression model which included sex, race, and age, levels only the factor of age had a significant effect on kallikrein levels. The p-value for the reduced model including only the factor of age was 0.0406 and the R-square was 0.038. Further analysis revealed that females did exhibit significantly higher kallikrein in individuals 40 years or older and that the effect of age appeared to be limited to females. It is concluded that both gender and age must be considered when evaluating salivary kallikrein changes in relationship to systemic disease.
Introduction
Glandular (tissue) kallikrein, a tryptic-like regulatory serine protease, is found in a variety of tissues and biological fluids including saliva [1]. Although specific functions have not yet been determined for this enzyme, it has been implicated in the regulation of local blood flow in salivary glands [2], the processing of polypeptide hormones such as epidermal growth factor [3], in ion transport in epithelial cells [4], and neutrophil chemotaxis [5]. Several groups of investigators have reported variations in salivary kallikrein as a function of systemic conditions. Decreases in salivary kallikrein
in patients with sarcoidosis without salivary gland involvement has been reported [6] as have perimenstrual increases [7]. Marked increases in tissue kallikrein in saliva upon restriction of dietary sodium has been observed [8]. This is similar to the changes observed for urinary tissue kallikrein [9] under similar physiological manipulation. Our laboratory has demonstrated higher levels of salivary kallikrein in subjects with tumors distant from the oral cavity when compared to control subjects [10]. More recently another group [11] has reported similar increases in tissue kallikrein in parotid saliva obtained from subjects with oral cancers.
30 In order to evaluate and extend these observations, more information is needed to define factors which might influence the level of salivary kallikrein in a normal population. For example, in the studies cited above, the potential relationship of salivary kallikrein levels and subject age was not directly addressed. Likewise, there is only limited information on the potential effect of sex and race on the levels of glandular kallikrein in saliva. Gender differences in urinary and serum levels of glandular kallikrein have been observed [12]. In this study, conducted under conditions of controlled dietary intake of sodium, females exhibited higher levels of total and active urinary kallikrein while males exhibited higher serum levels of immunoreactive kallikrein. Urinary excretion of kallikrein has been found to be higher in whites than in blacks [13]. The present study was designed to evaluate the effects of age, sex, and race on the level of salivary kallikrein in mixed saliva obtained from a normal population. The availability of such information would permit the use of saliva for large population screening studies such as the recent description of a relationship between urinary kallikrein levels and genetic predisposition to hypertension [14]. Methods and materials
D-ValylLeucylArginine-p-nitroanilide (S-2266) was a product of Kabi Laboratories, Stockholm, Sweden and was obtained from Helena Laboratories, Beaumont, Texas. All other chemicals were of at least reagent grade. Approximately 5 - 7 mL of whole saliva was collected from 114 individuals by paraffin-stimulation after the subjects had rinsed with deionized water. Collection time was 10 minutes or less. All subjects in the current study were seen between 0900 and 1200 to avoid possible complication arising from diurnal variation [15]. The saliva was centrifuged at 12,000 x g for 10 minutes at 4~ and the supernatant fractions stored in one mL portions at - 2 0 ~ for subsequent analysis. Salivary kallikrein was measured as previously described by this laboratory [16, 17]. Data is expressed as nmol substrate hydrolyzed per minute per mL saliva. Subjects were recruited from the patient pool in the University of North Carolina School of Dentistry. Since grouping by age was the primary focus for the current study, age was the criterion for subject selection. Each designated age group had
Agents Actions, 35 (1992) at least ten subjects. In the 20-34 year old young adult group, there were twenty-nine subjects reflecting both a greater age range and more available subjects in this group. Race and sex were noted and included in this analysis but neither was a criterion for subject selection. Because of this, there were more inconsistencies in subject distribution when viewed by sex or race. For example, there were more whites than blacks and more black females than black males. Three subjects were excluded from analyses. Two of these were American Indians and the third one, a 73 year old white male, exhibited an extreme outlier value for salivary kallikrein (29.9 nmol/min/mL). Information concerning health history and current medications was obtained from each subject. These items were grouped and utilized in the analyses to determine their potential effect on kallikrein levels. Kallikrein levels when expressed in the original units were not normally distributed. Values were transformed to natural logarithms for analyses. Regression analysis was used to assess effects of sex, race, and age on kallikrein levels. A stepwise regression was utilized to determine the effect of health and medication factors. The significance level for inclusion of these factors in the regression model was 0.15. Means and standard deviations were calculated according to sex, race, and age group. Unpaired t-test comparisons were made to assess differences attributable to sex or race and to assess differences attributable to sex in older subjects. Results
Distributions of mean kallikrein levels according to sex, race, and age group are shown in Tables 1 and 2. Differences attributable to race were not apparent. Kallikrein levels did appear to increase with age and to be higher in females than in males. A multiple regression analysis, with a model p-value of 0.0584 and R 2 value of 0.067105, showed that out of the factors of age, sex, and race, only the coefficient of age had a statistically significant p-value (0.0442). In reducing the regression model to the single factor of age, the R 2 value declined to 0.037928 (p=0.0406). The estimated equation obtained from this analysis was Log Kallikrein = 1.2606 + (0.00608 x age). Results of t-test comparisons according to race or sex were not signifi-
Agents Actions, 35 (1992)
31
Table 1
Table 4
Concentration of glandular kallikrein in human mixed saliva according to sex and race.
Categories of health and medication factors assessed and their frequency in the study population.
Mean kallikrein levels ~
Health condition ~
Male
Female
Black n=
4.56+3.56 8
5.73_+3.92 5.39• 20 28
White n=
4.64• 37
5.65+2.99 46
5.20• 83
Overall mean b n=
4.63 • 2.46 45
5.67 + 3.26 66
5,24 + 2.99 111
Frequency
Overall mean b
nmol substrate (S-2266) hydrolyzed per minute per mL mixed saliva. The assays were performed with 200 uL saliva and 800 uL 160 uM S-2266 in 50 mM Tris, pH 8.0 at 25~ Further description of assay conditions is contained in the text. b Differences according to sex or race were not significant using an unpaired t-test for comparison.
Table 2
Concentration of glandular kallikrein in human mixed saliva according to age group Age group
n
Mean kaUikrein levels
65
12 10 28 17 16 15 13
4.74+1.82 a 4.10___2.20 4.56___2.37 4.80+2.77 6.59_ 3.45 5,89--+3.23 6.30+4.34
111
5.24 __2.99
Total group
FreMedication quency
nmol substrate (S-2266) hydrolyzed per minute per mL mixed saliva. The assays were performed as described under Table I and in the text.
Table 3
Comparison of salivary kallikrein levels ~ in younger and older age groups. Age group
Males
Females
40 years n=
4.65• 19
6.87-t-3.71 b 32
Heart disease 8 High blood pressure 15 Cancer (not active) 13 Diabetes 4 Arthritis 2 Other 21
Anti-inflammatory Smooth muscle relaxant Antihypertensive Diuretic Antidepressants Other
9 4 13 12 2 13
a Past or present history of condition indicated. None of conditions included in the 'other' category constituted a distinct subgroup.
c a n t ( p = 0 . 8 0 7 9 a n d 0.1214, respectively). S i n c e t h e g e n d e r d i f f e r e n c e a p p r o a c h e d significance, t h e d a t a w a s r e - e x a m i n e d b y d i v i d i n g t h e g r o u p a t 40 years of age and evaluating differences in the y o u n g e r a n d o l d e r g r o u p s s e p a r a t e l y ( T a b l e 3). Mean values in the younger group were almost identical for males and females. In the older group, t h e m e a n level f o r m a l e s h a d n o t c h a n g e d a p p r e ciably, b u t t h e m e a n v a l u e f o r f e m a l e s h a d increased and was significantly higher than males. When males were excluded from the regression a n a l y s i s , t h e R 2 v a l u e f o r t h e single f a c t o r o f a g e w a s 0.1162 ( p = 0 . 0 0 5 1 ) c o n f i r m i n g t h a t a g e is a m u c h s t r o n g e r f a c t o r i n i n f l u e n c i n g k a l l i k r e i n levels i n f e m a l e s t h a n in m a l e s . I f f e m a l e s w e r e e x c l u d ed, t h e m o d e l w a s n o t s i g n i f i c a n t . D i s t r i b u t i o n o f h e a l t h a n d m e d i c a t i o n f a c t o r s in t h e s t u d y p o p u l a t i o n is s h o w n i n T a b l e 4. W h e n t h e s e f a c t o r s w e r e i n c l u d e d in a s t e p w i s e r e g r e s s i o n m o d e l a l o n g w i t h age, n o n e o f t h e f a c t o r s w e r e s i g n i f i c a n t i n i n f l u e n c i n g k a l l i k r e i n levels ( s e l e c t i o n p - v a l u e = 0.15 o r less). T h e n u m b e r o f p o s i t i v e s u b j e c t s i n t h e s u b g r o u p s e v a l u a t e d were, h o w e v e r , quite small and a consideration of their potential effect s h o u l d n o t b e e x c l u d e d i n f u t u r e studies. Discussion
a nmol substrate (S-2266) hydrolyzed per minute per mL of mixed saliva. Significantly higher than mean value for males >40 years of age, unpaired t-test, p=0.0191.
T h e r e s u l t s o f this s t u d y c l e a r l y i n d i c a t e a t e n d e n c y f o r k a l l i k r e i n levels to i n c r e a s e w i t h age i n f e m a l e s b u t n o t m a l e s . I n a p r e v i o u s s t u d y [12], h i g h e r u r i n a r y k a l l i k r e i n levels w e r e o b s e r v e d in f e m a l e s than in males. Subjects in that study, however, w e r e less t h a n 40 y e a r s o f age, w h e r e a s i n t h e p r e s ent study, gender differences were not significant
32 except in an older group. There were no differences in kallikrein levels attributable to race although such differences have previously been reported for urinary kallikrein [13]. It has been demonstrated that both human urinary and salivary kallikrein represent the same gene product [18, 19] based on nucleic acid sequence analysis but it is possible that there is tissue-specific expression such as that demonstrated in the rodent system [10]. An answer to this question will require the simultaneous measurement of tissue kallikrein in urine and saliva. There have only been limited studies on changes in specific proteins in saliva as a result of gender or aging. Most studies have been limited to the examination of possible changes in total protein concentration [21, 22]. However, there has been some suggestion of changes in specific proteins [23] and steroid hormones [24]. The present study demonstrates that there may be changes in selected proteins in saliva such as tissue kallikrein which are not acinar cell products [23] but rather a protein of striated duct cells [25]. It is noted that these are the testosterone-stimulated cells which in rodents are responsible for the sexual dimorphism in salivary tissue kallikrein expression [26]. Changing hormonal conditions in aging females may be a factor in the observations reported in the current study. These findings suggest that in studies evaluating salivary kallikrein, consideration must be given to a potential effect of age in female subjects and that possible gender differences should also be considered. However, with a consideration of such fac-tors, the use of saliva for the analysis of tissue kallikrein has considerable potential for use in large population studies [14].
Acknowledgements This work was supported in part by Grant DE-06997 from the National Institute of Dental Research. Received 27 December 1990; accepted by I. Ahnfelt-Ronne 19 June 1991
References [1] K. Mann, B. Lipp, J. Grunst, R. Geiger and H. J. Karl, Determination of kallikrein by radioimmunoassay in human body fluids. Agents Actions 10, 329-334 (1980).
Agents Actions, 35 (1992) [2] T. Berg, O. A. Carretero, A. G. Scicli, B. Tilley and J. W Stewart, Role of kinin in regulation of rat submandibulur gland blood flow. Hypertension 14, 73-80 (1989). [3] P. J. Issaksson, J. C. Dunbar and R. A. Bradshaw, Role of glandular kallikreins as growth factor processing enzymes: structural and evolutionary considerations. J. Cell. Biochem. 33, 65-75 (1987). 14] S. A. Lewis and W. P. Alles. Urinary kallikrein: aphysiological regulator y of epithelial ?Ca+- absorption. Proc. Natl. Acad. Sci. 83, 5345-5438 (1986). [5] T. Modeer, Human saliva kallikrein. Biological properties of saliva kallikrein isolated by use of affinity chromatography. Acta odont. Scand. 35, 23-29 (1977). [6] K. D. Bhoola, M. N. McNicol, S. Oliver and J. Foran, Changes in salivary enzymes in patients with sarcoidosis. New Eng. J. Med. 281, 877-879 (1969). [7] K. D. Bhoola, R. W. Matthews and E Roberts, A survey of salivary kallikrein and amylase in a population of schoolgirls throughout the menstrual cycle. Clin. Sci. Molec. Med. 55, 561-565 (1978). [8l D. Horwitz, D. Proud, W J. Lawton, K. N. Yates, P. Higher, J. J. Pisano and H. R. Keiser, Effects of restriction of sodium or administration of fludrocortisone on parotid salivary kallikrein in man. J. lab. clin. Med. 100, 146-154 (1982). [9] A. G. Scicli and O. A. Carretero, Renal kallikrein-kinin system. Kid. Int. 29, 120-130 (1986). [10] J. W. Jenzano, N. E Courts, D. A. Timko and R. L. Lundblad, Levels of glandular kallikrein in whole saliva obtained from patients with solid tumors remote from the oral cavity. J. dent. Res. 65, 67-70 (1986). [11] W H. Arnold, P. Bartholmes, H. Senkel, J. L. N. Roodenburg and R. Roder, Kallikrein activity in saliva of patients with inflammatory hyperplastic diseases of the oral mucous membrane. Adv. Exptl. Med. Biol. 247B, 385-387 (1989). [12] G. S. Hughes, Jr., H. S. Margolius, R. Peters, M. J. Oexmann, J. Chao and G. Lindenmayer, Gender differences of human tissue kallikrein and an erythrocyte kallikrein-like enzyme in essential hypertension. J. lab. clin. Med. 112, 612618 (1988). [13] D. Horwitz, H. S. Margolius and H. R. Keiser, Effects o f dietary potassium and race or urinary excretion o f kaltikrein and aldosterone in man. J. Clin. Endocrin. Metab. 47, 296299 (1978). [14] T. D. Berry, S. J. Hasstedt, S. C. Hunt, L. L. Wu, J. B. Smith, K. O. Ash, H. Kuida and R. R. Williams, A gene for high urinary kallikrein may protect against hypertension in Utah kindreds. Hypertension 13, 3 - 8 (1989). [15] J. W Jenzano, C. K. Brown, and S. M. Mauriello, Temporal variations in glandular kallikrein, protein and amylase in mixed human saliva. Archs. oral Biol. 32, 757-759 (1987). [161 J. W Jenzano, P. W. Daniel, R. T. Kent, J. L. Leal and D. L. Koth, Evaluation of kallikrein in human parotid and submandibular saliva. Archs. oral. Biol. 31, 627-628 (1986). [17] J. W Jenzano, J. C. Coffey, W. D. Heizer, R. L. Lundblad and A. G. Scicli, The assay of glandular kallikrein and prekallikrein in human mixed saliva. Archs. oral Biol. 33, 641-644 (1988). [18] D. Fukushima, N. Kitamura and S. Nakanishi, Nucleotide sequence of cloned cDNA for human pancreatic kallikrein. Biochemistry 24, 8037-8043 (1985). [19] B. A. Evans, Z. X. Yun, J. A. Close, G. W Tregear, N. Kitamura, S. Nakanishi, D. F. Callen, E. Baker, V. J. Hyland, G. R. Sutherland and R. I. Richards, Structure and
Agents Actions, 35 (1992) chromosomal localization of the human renal kallikrein gene. Biochemistry 27, 3124-3129 (1988). [20] P. L. Ashley and R. J. MacDonald, Tissue-specific expression of kallikrein-related genes in the rat. Biochemistry 24, 45204527 (1985). [21] H. Ben-Aryeh, A. Shaley, R. Szargel, A. Laor, D. Laufer and D. Gutman, The salivary flow rate and composition of whole and parotid resting and stimulated saliva in young and oM healthy subjects. Biochem. Med. and Metab. Biol. 36, 260-265 (1986). [22] C. W. Bales, J. H. Freeland-Graves, S. Askey, F. Behmardi, R. S. Pobocik, J. J. Fickel and P. Greenlee, Zinc, magnesium, copper, and protein concentrations in human saliva: age- and sex-related differences. Amer. J. Clin. Nutr. 51, 462-469 (1990).
33 [23] P. C. Fox, M. W. Heft, M. Herrera, M. R. Bowers, I. D. Mandel and B. J. Baum, Secretion ofantimicrobial proteins from the parotid glands of different aged healthy persons. J. Gerontol. 42, 466-469 (1987). [24] H. Ben-Aryeh, A. Aru-Nassar, D. Laufer, M. Lahav and Z. Shen-Ora, Salivary testosterone levels in men and women: changes with age and correlation with plasma testosterone. Israel J. Med. Sci. 25, 344-346 (1989). [25] R..1. MacDonald, H. S. Margolius and E. G. Erdos, Molecular biology of tissue kallikrein. Biochem. J. 253, 313-321 (1988). [26] S. R. Murray, J. Chao, F.-K. Lin and L. Chao, Kallikrein multigene families and the regulation of their expression. J. Cardiovascular Pharmacol. 15 (Suppl.), $7-S16 (1990).
0065-4299/92/020029-05 $1.50+0.20/0 9 1992 Birkh/iuser Verlag, Basel
The influence o f age, sex and race on salivary kallikrein levels in human mixed saliva Joyce W. Jenzano 1,2, Susan L. Hogan 1, and R.L. Lundblad 1,a,4 Dental Research Center ~and Departments of Dental Ecology ~ and Periodolatics 3, School of Dentistry and Departments of Pathology and Biochemistry 4, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7455 (USA)
Abstract
Variation in the level of salivary kallikrein in human saliva has been reported as a function of systemic conditions such as reduced salt intake and during the menstrual cycle. Higher levels of salivary kallikrein have been observed in subjects with tumors distant from the oral cavity when compared to control subjects. These studies have not evaluated factors, such as age, which might influence the concentration of glandular kallikrein in saliva. The purpose of the present study was to determine the variation of salivary kallikrein concentration as a function of age. Differences attributable to sex or race were also evaluated. Mixed saliva was collected from 114 subjects, ages 5-91, by paraffin stimulation. Samples were centrifuged and stored at - 20 ~ for subsequent analysis. Glandular kallikrein activity was assayed using D-ValylLeucylArginine-p-nitroanilide as the substrate. In a linear regression model which included sex, race, and age, levels only the factor of age had a significant effect on kallikrein levels. The p-value for the reduced model including only the factor of age was 0.0406 and the R-square was 0.038. Further analysis revealed that females did exhibit significantly higher kallikrein in individuals 40 years or older and that the effect of age appeared to be limited to females. It is concluded that both gender and age must be considered when evaluating salivary kallikrein changes in relationship to systemic disease.
Introduction
Glandular (tissue) kallikrein, a tryptic-like regulatory serine protease, is found in a variety of tissues and biological fluids including saliva [1]. Although specific functions have not yet been determined for this enzyme, it has been implicated in the regulation of local blood flow in salivary glands [2], the processing of polypeptide hormones such as epidermal growth factor [3], in ion transport in epithelial cells [4], and neutrophil chemotaxis [5]. Several groups of investigators have reported variations in salivary kallikrein as a function of systemic conditions. Decreases in salivary kallikrein
in patients with sarcoidosis without salivary gland involvement has been reported [6] as have perimenstrual increases [7]. Marked increases in tissue kallikrein in saliva upon restriction of dietary sodium has been observed [8]. This is similar to the changes observed for urinary tissue kallikrein [9] under similar physiological manipulation. Our laboratory has demonstrated higher levels of salivary kallikrein in subjects with tumors distant from the oral cavity when compared to control subjects [10]. More recently another group [11] has reported similar increases in tissue kallikrein in parotid saliva obtained from subjects with oral cancers.
30 In order to evaluate and extend these observations, more information is needed to define factors which might influence the level of salivary kallikrein in a normal population. For example, in the studies cited above, the potential relationship of salivary kallikrein levels and subject age was not directly addressed. Likewise, there is only limited information on the potential effect of sex and race on the levels of glandular kallikrein in saliva. Gender differences in urinary and serum levels of glandular kallikrein have been observed [12]. In this study, conducted under conditions of controlled dietary intake of sodium, females exhibited higher levels of total and active urinary kallikrein while males exhibited higher serum levels of immunoreactive kallikrein. Urinary excretion of kallikrein has been found to be higher in whites than in blacks [13]. The present study was designed to evaluate the effects of age, sex, and race on the level of salivary kallikrein in mixed saliva obtained from a normal population. The availability of such information would permit the use of saliva for large population screening studies such as the recent description of a relationship between urinary kallikrein levels and genetic predisposition to hypertension [14]. Methods and materials
D-ValylLeucylArginine-p-nitroanilide (S-2266) was a product of Kabi Laboratories, Stockholm, Sweden and was obtained from Helena Laboratories, Beaumont, Texas. All other chemicals were of at least reagent grade. Approximately 5 - 7 mL of whole saliva was collected from 114 individuals by paraffin-stimulation after the subjects had rinsed with deionized water. Collection time was 10 minutes or less. All subjects in the current study were seen between 0900 and 1200 to avoid possible complication arising from diurnal variation [15]. The saliva was centrifuged at 12,000 x g for 10 minutes at 4~ and the supernatant fractions stored in one mL portions at - 2 0 ~ for subsequent analysis. Salivary kallikrein was measured as previously described by this laboratory [16, 17]. Data is expressed as nmol substrate hydrolyzed per minute per mL saliva. Subjects were recruited from the patient pool in the University of North Carolina School of Dentistry. Since grouping by age was the primary focus for the current study, age was the criterion for subject selection. Each designated age group had
Agents Actions, 35 (1992) at least ten subjects. In the 20-34 year old young adult group, there were twenty-nine subjects reflecting both a greater age range and more available subjects in this group. Race and sex were noted and included in this analysis but neither was a criterion for subject selection. Because of this, there were more inconsistencies in subject distribution when viewed by sex or race. For example, there were more whites than blacks and more black females than black males. Three subjects were excluded from analyses. Two of these were American Indians and the third one, a 73 year old white male, exhibited an extreme outlier value for salivary kallikrein (29.9 nmol/min/mL). Information concerning health history and current medications was obtained from each subject. These items were grouped and utilized in the analyses to determine their potential effect on kallikrein levels. Kallikrein levels when expressed in the original units were not normally distributed. Values were transformed to natural logarithms for analyses. Regression analysis was used to assess effects of sex, race, and age on kallikrein levels. A stepwise regression was utilized to determine the effect of health and medication factors. The significance level for inclusion of these factors in the regression model was 0.15. Means and standard deviations were calculated according to sex, race, and age group. Unpaired t-test comparisons were made to assess differences attributable to sex or race and to assess differences attributable to sex in older subjects. Results
Distributions of mean kallikrein levels according to sex, race, and age group are shown in Tables 1 and 2. Differences attributable to race were not apparent. Kallikrein levels did appear to increase with age and to be higher in females than in males. A multiple regression analysis, with a model p-value of 0.0584 and R 2 value of 0.067105, showed that out of the factors of age, sex, and race, only the coefficient of age had a statistically significant p-value (0.0442). In reducing the regression model to the single factor of age, the R 2 value declined to 0.037928 (p=0.0406). The estimated equation obtained from this analysis was Log Kallikrein = 1.2606 + (0.00608 x age). Results of t-test comparisons according to race or sex were not signifi-
Agents Actions, 35 (1992)
31
Table 1
Table 4
Concentration of glandular kallikrein in human mixed saliva according to sex and race.
Categories of health and medication factors assessed and their frequency in the study population.
Mean kallikrein levels ~
Health condition ~
Male
Female
Black n=
4.56+3.56 8
5.73_+3.92 5.39• 20 28
White n=
4.64• 37
5.65+2.99 46
5.20• 83
Overall mean b n=
4.63 • 2.46 45
5.67 + 3.26 66
5,24 + 2.99 111
Frequency
Overall mean b
nmol substrate (S-2266) hydrolyzed per minute per mL mixed saliva. The assays were performed with 200 uL saliva and 800 uL 160 uM S-2266 in 50 mM Tris, pH 8.0 at 25~ Further description of assay conditions is contained in the text. b Differences according to sex or race were not significant using an unpaired t-test for comparison.
Table 2
Concentration of glandular kallikrein in human mixed saliva according to age group Age group
n
Mean kaUikrein levels
65
12 10 28 17 16 15 13
4.74+1.82 a 4.10___2.20 4.56___2.37 4.80+2.77 6.59_ 3.45 5,89--+3.23 6.30+4.34
111
5.24 __2.99
Total group
FreMedication quency
nmol substrate (S-2266) hydrolyzed per minute per mL mixed saliva. The assays were performed as described under Table I and in the text.
Table 3
Comparison of salivary kallikrein levels ~ in younger and older age groups. Age group
Males
Females
40 years n=
4.65• 19
6.87-t-3.71 b 32
Heart disease 8 High blood pressure 15 Cancer (not active) 13 Diabetes 4 Arthritis 2 Other 21
Anti-inflammatory Smooth muscle relaxant Antihypertensive Diuretic Antidepressants Other
9 4 13 12 2 13
a Past or present history of condition indicated. None of conditions included in the 'other' category constituted a distinct subgroup.
c a n t ( p = 0 . 8 0 7 9 a n d 0.1214, respectively). S i n c e t h e g e n d e r d i f f e r e n c e a p p r o a c h e d significance, t h e d a t a w a s r e - e x a m i n e d b y d i v i d i n g t h e g r o u p a t 40 years of age and evaluating differences in the y o u n g e r a n d o l d e r g r o u p s s e p a r a t e l y ( T a b l e 3). Mean values in the younger group were almost identical for males and females. In the older group, t h e m e a n level f o r m a l e s h a d n o t c h a n g e d a p p r e ciably, b u t t h e m e a n v a l u e f o r f e m a l e s h a d increased and was significantly higher than males. When males were excluded from the regression a n a l y s i s , t h e R 2 v a l u e f o r t h e single f a c t o r o f a g e w a s 0.1162 ( p = 0 . 0 0 5 1 ) c o n f i r m i n g t h a t a g e is a m u c h s t r o n g e r f a c t o r i n i n f l u e n c i n g k a l l i k r e i n levels i n f e m a l e s t h a n in m a l e s . I f f e m a l e s w e r e e x c l u d ed, t h e m o d e l w a s n o t s i g n i f i c a n t . D i s t r i b u t i o n o f h e a l t h a n d m e d i c a t i o n f a c t o r s in t h e s t u d y p o p u l a t i o n is s h o w n i n T a b l e 4. W h e n t h e s e f a c t o r s w e r e i n c l u d e d in a s t e p w i s e r e g r e s s i o n m o d e l a l o n g w i t h age, n o n e o f t h e f a c t o r s w e r e s i g n i f i c a n t i n i n f l u e n c i n g k a l l i k r e i n levels ( s e l e c t i o n p - v a l u e = 0.15 o r less). T h e n u m b e r o f p o s i t i v e s u b j e c t s i n t h e s u b g r o u p s e v a l u a t e d were, h o w e v e r , quite small and a consideration of their potential effect s h o u l d n o t b e e x c l u d e d i n f u t u r e studies. Discussion
a nmol substrate (S-2266) hydrolyzed per minute per mL of mixed saliva. Significantly higher than mean value for males >40 years of age, unpaired t-test, p=0.0191.
T h e r e s u l t s o f this s t u d y c l e a r l y i n d i c a t e a t e n d e n c y f o r k a l l i k r e i n levels to i n c r e a s e w i t h age i n f e m a l e s b u t n o t m a l e s . I n a p r e v i o u s s t u d y [12], h i g h e r u r i n a r y k a l l i k r e i n levels w e r e o b s e r v e d in f e m a l e s than in males. Subjects in that study, however, w e r e less t h a n 40 y e a r s o f age, w h e r e a s i n t h e p r e s ent study, gender differences were not significant
32 except in an older group. There were no differences in kallikrein levels attributable to race although such differences have previously been reported for urinary kallikrein [13]. It has been demonstrated that both human urinary and salivary kallikrein represent the same gene product [18, 19] based on nucleic acid sequence analysis but it is possible that there is tissue-specific expression such as that demonstrated in the rodent system [10]. An answer to this question will require the simultaneous measurement of tissue kallikrein in urine and saliva. There have only been limited studies on changes in specific proteins in saliva as a result of gender or aging. Most studies have been limited to the examination of possible changes in total protein concentration [21, 22]. However, there has been some suggestion of changes in specific proteins [23] and steroid hormones [24]. The present study demonstrates that there may be changes in selected proteins in saliva such as tissue kallikrein which are not acinar cell products [23] but rather a protein of striated duct cells [25]. It is noted that these are the testosterone-stimulated cells which in rodents are responsible for the sexual dimorphism in salivary tissue kallikrein expression [26]. Changing hormonal conditions in aging females may be a factor in the observations reported in the current study. These findings suggest that in studies evaluating salivary kallikrein, consideration must be given to a potential effect of age in female subjects and that possible gender differences should also be considered. However, with a consideration of such fac-tors, the use of saliva for the analysis of tissue kallikrein has considerable potential for use in large population studies [14].
Acknowledgements This work was supported in part by Grant DE-06997 from the National Institute of Dental Research. Received 27 December 1990; accepted by I. Ahnfelt-Ronne 19 June 1991
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