Changes in protein and glycoprotein concentrations in human submaxillary saliva under various stimulatory conditions

.4rch. oral Bfol. Vol.11. pp.437449,

CHANGES TRATIONS

1966. Pergamon Press Ltd.

Printed inGt.Britain.

IN PROTEIN AND GLYCOPROTEIN CONCENIN HUMAN SUBMAXILLARY SALIVA UNDER VARIOUS STIMULATORY CONDITIONS R. C. CALDWELLand W. PIGMAN

Departments of Oral Biology and Biochemistry, University of Alabama Medical Center, Birmingham, Alabama, U.S.A. Summary-The concentrations of protein and protein-bound carbohydrates in human submaxillary saliva depended on salivary flow rate and not on the specific type of gustatory stimulus. “Unstimulated” saliva had the highest concentration of protein-bound carbohydrates. Low flow rates were associated with the lowest concentrations of protein and protein-bound carbohydrates, and as flow rate was increased, there was an accompanying increase in the concentrations of these substances. The concentrations of protein-bound carbohydrates were highly variable in “unstimulated” saliva. Stimulated saliva was relatively more predictable in composition. Protein concentration averaged 122 f 76 (S.D.) mg %; however, the change in concentration from the lowest to the highest value recorded represented an increase of 1600%. Similarly, the concentration of protein-bound carbohydrates varied as much as 450% in the case of galactose. Average concentrations (PM) for protein-bound carbohydrates were: galactose, 395 i 235; hexosamine, 290 i 169 ; sialic acid, 151 f 83 : fucose, 160 f 108. Secretors of blood group substances had concentrations of protein-bound carbohydrates higher than non-secretors, except for sialic acid concentration which was similar for the two groups. The sialic acid/fucose ratio for secretors was 064 f 0.12 and did not vary with flow rate. The ratio for non-secretors was l-40&0*35 and rose with increasing flow rate. THE LITERATURE pertaining

to the concentration of protein in human saliva is con fusing. Some authors (BRAMKAMP,1936; ARAKI, 1951) state that the protein concentration rises with flow rate, while others claim that, although variable, the protein concentration is unrelated to flow rate (TENENBAUMand KARSHAN, 1939; DEAKINSer al., 1941; FERGUSON,KRAHN and HILDES, 1958). Some of the confusion may be due to differences in the type of saliva involved, i.e., whether whole, parotid, or submaxillary. Generally, however, too little attention has been paid to the possible effect of the conditions under which the saliva was collected. The volume of saliva collected varies in different reports, and in some cases care was not taken to void a preliminary sample prior to collecting the secretion elaborated during the stimulatory condition under study. In some studies the measurements of protein concentration were not made over the full range of salivary flow rates which can occur under physiological conditions. For example, a relationship between parotid salivary flow rate and parotid amylase activity has been described by DAWES and JENKINS(1964) who showed that the rates of flow recorded in previous studies (SCHNEYER, 1956; NEWBRUN, 1962) were too low to observe the relationship which exists. Also, the effects of different gustatory or other 437

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R. C. CALDWELL ANDW. PIGMAN

types of stimuli on the protein concentration in submaxillary saliva have not been investigated. The glycoproteins of human submaxillary saliva have been studied by MANUEL and ELLISON(1963); MANDEL, THOMPSONand ELLISON(1963, 1964); CALDWELL, (1964) ; CALDWELLand PIGMAN(1964,1965a and b). However, apart from our previous work; no attention had been given to the relationship between salivary flow rate and the concentration of glycoproteins in the submaxillary saliva of individual subjects. The foregoing types of observations prompted this study of the changes in the protein and glycoprotein components of human submaxillary saliva under various stimulatory conditions. Among the variables considered were salivary flow rate, type of stimulation used to elicit salivary flow, sample volume, individual variations in different subjects, and day-to-day variations in the same subject. The blood group substance secretor status was also taken into consideration, because previous reports (CALDWELL,1964; CALDWELLand PIGMAN,1964, 1965b) have shown that the composition of the human submaxillary glycoproteins depends on the secretor status of the individual. MATERIALS AND METHODS Nine apparently healthy, male dentists or dental students, ranging in age from 20 to 40 years, took part in the study. Secretor status determinations by the method of KABAT(1965, p. 56), revealed that five of the subjects were non-secretors of blood group substances, and four were secretors. Blood typing at the University of Alabama Blood Bank showed that all the subjects who had been classified as secretors had Lewis (b) substance in their blood, but did not have Lewis (a) substance. This is the usual relationship between secretor status and Lewis blood type (KABAT, 1956, p. 14). The non-secretors were all Lewis (b) negative, and four of the five were Lewis (a) positive. The fifth non-secretor had neither of the Lewis substances in his blood. Although it is unlikely that a common duct for the sublingual and submaxillary glands is present in humans, sialograms were obtained for subject 1, whose saliva was used most often, by X-raying the gland on each side following injection down each duct of a radio-opaque solution (Pantopaque). The roentgenograms revealed a single main duct leading from each submaxillary gland with no anastomoses to the sublingual glands on either side. For the other subjects a close visual inspection of the floor of the mouth was made to identify the area to be covered by the collection chamber of the apparatus. The appliances for collection of the salivary secretion were constructed individually for each subject, according to the method of SCHNEYER(1955). Submaxillary secretion was stimulated to flow from both the submaxillary glands under the influence of the chosen gustatory stimulus, and after the first 2 ml were voided, the secretion was collected in a glass graduated cylinder packed in ice. Throughout the period of collection and subsequent dialysis, the temperature of the sample was kept at 2-4”C. Four types of gustatory stimulation were represented by aqueous solutions of 30 “/, sodium chloride (salt), 0.15 % quinine HCl (bitter), 40% sucrose (sweet), and 2% citric acid (sour). The concentrations of the solutions were chosen in accordance-with

HLIMAN SALIVARY PROTEI% AM) GLYCOPROTEIK COXENTRATIONS

439

the observations of C~~AUNCEY and SHANNON(1960), who reported the concentrations which elicit maximal secretion from the human parotid gIand. The solution was sprayed on the tongue at 30-set intervals, using an atomizer (DeVilbiss No. 15) to minimize mechanical stimulation such as that produced by swabbing the tongue. Under the, conditions described here, approximately O-8ml of the stimulatory solution was applied to the tongue each minute. On various occasions more dilute solutions were used to give lower salivary flow rates. In some cases water was sprayed on the tongue for a comparison with the other stimulatory agents. “Unstimulated” secretion was collected whilst the subject rested almost asleep in a chair for as long as I hr. Combined physical and gustatory stimulation was afforded in some instances by sucking or chewing lime-flavoured candies. The time required for the collection of each sample was recorded with a stop watch. Fifteen milliliters were usually collected, because the chemical anaIytica1 methods required this volume. Aliquots for each determination were drawn from the accumulated volume of secretion rather than by collection of serial samples for each determination. This was desirable in view of variations which were observed in the hexosamine concentrations of 2 ml saliva samples collected on six successive occasions during a period of 30 min (CALDWELL,1964). For instance, in dialysed submaxillary secretion at 0,4, 8, 12, 18 and 30 min after beginning with 2% citric acid stimulation, the hexosamine concentrations were 210,325,375,380,355 and 400 PM. To check for the presence of bacterial contamination, the separated secretion was collected at different times and plated on nutrient and blood agar. In the opinion of a consultant microbioiogist (Dr. Charles Winkler), the secretion appeared to be free from bacterial contamination. The chemical analytical methods used were identical to those described in a previous paper (CALDWELLand PIGMAN,1965b) and were carried out following dialysis of the samples against distilled water at 2-4”C for 18 hours. The methods were: for hexose, Dische’s cysteine-sulphuric acid method with a galactose standard (DISCHE, 1955): for fucose, DISCHE’Scysteine-sulphuric acid method (CyR-IO) with a fucose standard (DISCHE and SHETTLES,1948); for hexosamine, Boas’ modification of the ElsonMorgan method, with a glucosamine HCl standard (BOAS, 1953); for sialic acid, the direct Ehrlich with a standard of N-acetylneuraminic acid (PIGMANet ul., 1958); for protein, a micro-Kjeldahl method (CLARK,1943), using the factor of 6.25 for conversion to protein. The protein nitrogen was corrected for the nitrogen content of the sialic acid and hexosamine present in the dialysed secretion. In every instance, the sample from one individual was analysed separately without resort to pooling of saliva from different individuals or from the same subject on different occasions. Saliva was never collected from an individual more than once a day. The chemical analyses were first carried out on a large number of occasions on submaxillary secretion from two subjects. Subject 1 was a male non-secretor of bfood group substances; subject 2 was a maIe secretor. The findings on these two were then confirmed by study of four more male non-secretors (subjects 3-6) and three more male secretors (subjects 7-9). In subsequent work to investigate special points, it was confirmed further that the finding to be described were consistently observed.

R. C. CALDWELL AND W.

440

PIGMAN

RESULTS

Efect offlow rate on submaxillary protein concentration

The concentration of protein in the submaxillary saliva of nine individuals is shown in Fig. 1. Four of the subjects were secretors of blood group substances and five were non-secretors. For both groups, a substantial increase in protein concentration 350

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rate on the concentration of protein in human submaxillary

saliva. occurred as the flow rate increased. For instance, in the case of one subject, on fortyeight separate occasions when saliva was collected from the submaxillary glands, the flow rate ranged from 0.09 to 3.28 ml/mm per gland pair. Over this thirty-five fold increase in flow rate, a large increase in the concentration of protein occurred. Expressed as percentage increase, the change in protein concentration from lowest (18 mg %) to highest (300 mg %) values for the submaxillary secretion of this individual was almost 1600 %. Sufficient data were available for individual subjects to demonstrate that the increase in protein concentration was not linear, but first decreased slightly and then increased. On occasions when the secretory rate was very slow (&‘unstimulated”), the concentration of total protein was higher than for mild stimulatory conditions. The lowest concentrations were for mild stimulatory conditions. At fast flow rates the submaxillary protein concentration for all subjects reached the highest values. Protein values higher than would be expected from the general trend occurred in secretions stimulated by 30 % NaC 1. It had been reported that successive samples of parotid saliva in the dog or cat contain progressively smaller amounts of protein (ANREP, 1921; STAVRAKY, 1940).

HLTMAN SALIVARY PROTEIN AND GLYWPROTEIN

441

CONCENTRA~ONS

To check for this possibility in human submaxillary secretion, serial samples of saliva were collected separately from two subjects and analysed for total nitrogen and nondialysable nitrogen. The results of these experiments (Fig. 2) show that no decrease in

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the protein nitrogen content of saliva took place over a period of 30-40 min when the flow rate was kept fairly constant. In both subjects the first sample contained less nitrogen than subsequent samples. Two millilitres were discarded on all other occasions prior to collecting the secretion for chemical analysis. The somewhat higher fluctuations in experiment 2 were attributable to di~c~ties in maint~ning a steady flow of saliva. The salivary flow rates (ml~min per gland pair) for this experiment for successive samples were l-69,2*06, 1*94,2-36,1*94,2-24,2*07 and 2-41. In experiment 1, the flow rates were 0.97,0=97,0,99, i.00, 1.00 and 0.95 mI/min per gland pair. &j’tict qfJIow rutr on the protein-hound carbohydrate ~Jsubmxillary secretion

Figure 3 shows the variation of the concentration of protein-bound hexose in subject at different salivary flow rates. It can be seen that “unstimulated” submaxillary saliva had the highest concentration of hexose (824 FM) but that on an occasion when the flow rate was very low (O-15 mlimin) with the sucrose stimulus, the concentration of bound hexose was high. As with the protein concentration, the lowest carbohydrate concentrations occurred under conditions of mild stimulation, In turn, water, sucrose, quinine, salt, citric acid, and lime candy with chewing caused increasingly faster flow rates and an increasingly greater hexose content per unit volume of safiva. On several occasions, the gustatory stimulus was varied by using different 1 (non-secretor)

442

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Fro. 3. Effect of flow rate on the concentration of protein-bound hexose in human submaxillarysaliva. concentrations of the same material and, as the solution was more dilute, the flow rate decreased, and the concentration of bound hexose fell. For example, when citric acid was used at 0.02% concentration instead of 2% concentration, a flow rate of 051 mI/min per gland pair was observed instead of 1.07-1.80 mllmin per gland pair. At both the low and high flow rates, the concentration of hexose observed followed the general pattern of the curve (Fig. 3). Thus, the concentration of protein-bound hexose depended on flow rate over the range of flow rates recorded. For subject 1, the increase in hexose concentration in stimulated saliva was about 250 % from the lowest (149 FM) to the highest (530 ,uM) hexose value. The greatest difference (450 %) observed in hexose concentrations was between unstimulated saliva (824 PM) and saliva flowing slowly (149 PM). The application of a dilute solution of sodium chloride (0.3 %) resulted in a secretion with a hexose concentration which followed the curve, but more concentrated sodium chloride (30%) produced a secretion with a hexose concentration which was greater that thegeneral pattern. This effect of salt in high concentration was also observed with hexosamine, sialic acid, and fucose concentrations in saliva and, as mentioned earlier, in the case of subject 1 was especially marked for protein concentration. Another interesting finding was that, in subject 1, most of the hexose values obtained for samples collected under the influence of sucking lime candy seemed lower than the general trend. The same was true for hexosamine and fucose, but not sialic acid values. However, since this deviation was not generally observed in the other eight subjects, the curve on Fig. 3 was drawn without regard to the low values recorded while sucking the lime candy. Nevertheless, since preliminary evidence suggests a similar effect on parotid secretion in another subject, it should be mentioned at this time.

HUMAN

SALNARY

PROTEX-4 AN0

GLYOX’ROTEW

CONCE~ATIONS

442

One factor ofpossible significance in relation to the low carbohydrate values obtained by sucking is illustrated in Fig. 4 (PLATE1). This figure gives the drop pattern of the secretions under different stimulatory conditions. The salivary flow rate, measured in drops per minute, increased from 12 drops per minute with the sucrose stimulation to over 60 per minute for the stimulation caused by chewing lime candy. However, the stimulation induced by sucking lime candy caused an uneven flow of saliva with flurries of drops separated by intervals of no secretion. In a general way, the other five flow patterns were less uneven and could be described as steady rather than intermittent, as was the case with sucking. The periods of non-secretion with sucking coincided with the act of sucking, and after each sucking action the flurries of drops occurred. Visual observation of other subjects also showed that the intermittent type of secretion often took place during sucking. If the hexose values are evaluated on the basis of the output of bound hexose per minute versus flow rate, the relationship in Fig. 5 is obtained. Over collection periods varying from 4-6 to 71.0 min, a smoothly increasing output per minute resulted as the 8.0

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fiow rate increased. No fatigue effect was noticed on the carbohydrate concentration at the recorded flow rates, and, although the usual volume collected during this phase of the work was 15-l 6 ml, the described relationships still held when the volume collected at one time was increased to SO-60 ml. However, it was usually not possible to maintain the highest flow rates for a time sufficient to collect 50-60 ml samples. Figures 6 and 7 show composite graphs prepared by using a “moving average” method, in which the con~ntrations for each flow rate interval of O-2 mllmin were averaged. For example, in Fig. 6, five hexose concentrations were available for the

444

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interval of OGO-0.19 ml/m& and these were averaged to give a value of 0.53 PM for an average flow rate of 0.15 ml/min. Data obtained in this fashion gave the relationships seen in Figs. 6 and 7, which show the relative concentrations of protein-bound hexose, hexosamine, sialic acid, and fucose at various salivary flow rates for a non-secretor and a secretor of blood group substances. The hexose concentration always was higher than hexosamine, which in turn was greater than that of sialic acid or fucose. Figure

445

HUMAN SALNARY PROTEIN AND GLYCOPROTEIN CONCENTRATIONS

6 shows that for non-secretor saliva, the sialic acid was in higher concentration than fucose except at low flow rates, at which sialic acid and fucose were in similar concentrations. In contrast, Fig. 7 reveals the reverse of this situation for secretor saliva, for which the fucose was always at a higher concentration than the sialic acid. The results obtained by analysis of the submaxillary saliva from the additional seven subjects confirmed the observations made for subjects 1 and 2. The sialic acid: fucose ratios for secretors ranged from 0.40 to 0.90 with an average value of 0~64f O-12 (SD.). The sialic acid: fucose ratios ranged from 0~86 to 2.44 and averaged l-40 :t 0.35 tS,D,) for non-secretors. For non-secretors, the sialic acid: fucose ratio increased as salivary flow rate increased, whereas for secretors the ratio was fairly constant. Figure 8 illustrates this difference and compares the data for all nine subjects. It is apparent that submaxillary

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Frc;. 8. Effect of flow rate on the sialic acidjfucose ratio in submaxiI1at-y saliva from secretors and non-secretors of blood group substances.

salivas of secretors and non-secretors are similar in several respects, but differ in the relative proportions of sialic acid and fucose. The difference is apparent at all salivary flow rates and particularly obvious at high flow rates. Table 1 includes the ranges, means, and standard deviations of the concentrations of the protein and non-dialysable carbohydrates in human submaxillary saliva. Total protein averaged 122-76 mg/lOO ml, with no difference between the secretor and non\ecretor groups. The average concentrations of galactose, hexosamine, and fucose were higher for the secretor than for the non-secretor group. The student “t” test showed that these were statistically significant differences at the O-01 level, but the differences

446

R.C.

W. PIGMAN

CALDWELLAND

TABLE~.CONCENTRAI.I~NSOF'PROTEIPL'ANDNON-DIALYSABLECARBOHYDRATESMHUMANSUBMAXILLARY SALIVA

._._

All subjects* Range Mean IS.D. Protein (mg~lOOm1) Glactose (PM) Hexosamine (PM ) Sialic Acid (FM) Fucose (PM)

18-348 149-1879 127-1659 52-733 48-670

Secretors Range Mean &S.D. 29-267 223-970 158-643 56-331 103-434

122i76 3955235 290+169 151&83 16Oi108

-___

-.._

Non-secretors Range Mean &SD.

119&62 485f177 352&102 168&70 259579

124*t86 344+249 254*190 142185 104579

18-348 149-1879 127-1659 52-733 48-670

* Submaxillary saliva from nine male subjects was ana.lysed on 130 occasions. Four of the subjects Submaxillary salivary gland were secretors, and five were non-secretors of blood group substances. flow rates ranged from 0.24 to 3.97 ml/min per gland pair for secretors and from 0.05 to 3.78 ml/mm per gland pair for non-secretors.

in sialic acid concentrations were not significant. These data extend the data published earlier by us (CALDWELL and PIGMAN,. 1965b) in which only one type of stimulus was used and the range of flow rates recorded was considerably narrower. Effect of gustatory stimuli on saliva compositiorl

Although a variety of types of stimuli was used to elicit salivary flow, the concentration of protein and glycoprotein was affected less by the nature of the stimulus itself than by the rate of flow of saliva produced by the stimulus. The average concentrations and flow rates for each of seven different stimulatory conditions are shown in Fig. 9. It seems clear that the various carbohydrates changed in concentration according to the flow rate caused by the different stimuli, whether they were gustatory stimuli sprayed on the tongue, water, or the chewing of a lime SUBJECT

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TYPES

OF

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1

2 3 4

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of protein-bound

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HUMAN

SALIVARY

PROTEIN

AND

GLYCOPROTEIN

CONCENTRATIONS

447

Aavoured candy. However, sodium chloride at 30 “/dconcentration did not follow the usual pattern and caused the secretion of a saliva comparatively richer in protein-bound carbohydrate (Fig. 9). As mentioned earlier, the total protein concentrations were also elevated disproportionately by the 30% sodium chloride gustatory stimulus.

DISCUSSION

The results of this study emphasize and extend our previous observations (CALDWELL and PIGMAN,1965b), which demonstrated the need for cautious interpretation of the significance of variations in protein or glycoprotein concentrations in human subm~i~ary saliva. It is not only impo~ant to know the subject’s blood group secretor status, but also the flow rate of the saliva. For instance, the fact that non-di~ysable sialic acid was in different concentration in submaxillary saliva from males and females ( MANDELef uJ., 1964) should not yet be taken as evidence for a sex difference until it is shown that this difference exists when the groups are balanced with respect to salivary flow rate and secretor status. Generally, pooling saliva from different subjects would seem to be inadvisable in future attempts to study differences in saliva composition in health or disease. As has been reviously discussed (CALDWELLand PICMAN,1965b), the non-dialysable carbohydrates are presumably covalently bonded to protein as in other animal glycoproteins. The range of concentration of these glycoproteins reported then was for one type of stimulation (sucking lime candy). The data presented in Table 1 extend these ranges of concentration for a variety of stimulatory conditions and demonstrate the wide physiological variations in protein and glycoprotein content of human submaxillary saliva. Normal variations of 1600 % in protein concentration and 450 “/d. in protein-bound carbohydrate concentration make it difficult to make meaningful comparisons of groups of subjects. Since “unstimulated” saliva varies tremendously in glycoprotein content, salivas should be obtained by a reproducible stimulus. Under such conditions, a roughly predictable concentration exists for healthy individuals from time to time, but the actual values are related to the flow rate of the secretion. Thus, the “standard curve” of concentration versus flow rate for an individual should be determined before considering whether or not a particular concentration constitutes a significant deviation from the normal. The strong salty stimulus gave disproportionately high values, as had previously been shown for parotid saliva by DAWES and JENKINS (1964). Also, the action of sucking could cause low values. With sucking, the pattern of flow of the secretion was not continuous, but it is not known if this is the reason for the low concentrations of protein-bound hexose, hexosamine, and fucose in one of our experimental subjects. The ratios of sialic acid to fucose in the submaxillary saliva of non-secretors of blood group substances increased as the flow rate increased. It has been previously shown that several glycoproteins are present in human submaxilfary secretion (MANDEL. and ELLISON,1963 ; CALDWELLand PIGMAN, 1965a), and the increase in sialic acid :

fucose ratios with flow rate observed in this study suggests that the relative proportions of the various glycoprotein components of the saliva vary with Bow rate, at feast in the

448

R. C.

CALDWELL ANDW. PI~MAN

case of non-secretors of blood group substances. Also, the relative amounts of total carbohydrate to total protein decrease as flow rate increases. To our knowledge, it has not been previously recognized that a relation exists between flow rate and protein and glycoprotein concentrations in human submaxillary saliva. It is interesting that, especially for the glycoproteins, the relationship with flow rate should be U-shaped. BURGEN(1956) has shown that the U-shaped dependence on flow rate in the case of potassium in small samples of dog submaxillary secretion is eliminated as the size of the sample was increased. This was not the case in the present study. The basis for the increase in concentration of the salivary components with flow rate is obscure. BURGENand EMMELIN(1961) have suggested that the relationship is actually not between protein output and flow rate, but is a function of the stimulation of the gland. Acknowledgement-This investigation was supported by U.S.P.H.S. grant No. DE-01858 from the National Institute of Dental Research, and part of the work was performed under a research career development award No. GM-K3-15,280. The authors wish to thank Mrs. FRANCESW. WELLSfor valuable technical assistance. R6snm&-Les concentration en proteines et en hydrates de carbone, lies aux proteines, de salive humaine sous-maxillaire depend de la vitesse de &c&ion salivaire et non du type particulier de stimulus gustatif utilid. La salive “non stimulte” prbente la concentration la plus Clevee en hydrates de carbone, lies aux proteines. Des vitesses de s&c&ion lentes sont assock& avec les plus faibles concentrations de prottines et d’hydrates de carbone, lies aux prot6ines. Lorsque la vitesse de secretion est plus &levee, il n’y a pas d’augmentation concomitante des concentrations de ces substances. Les concentrations des hydrates de carbone, lies aux proteines sont hautement variables dans la salive “non stimul&e”. La salive stimulee est relativement plus stable en composition. La concentration en proteine avoisine 122 & 76 (D.S.) mg%; cependant la difference en concentration de la valeur la plus basse a la plus Blev&erepresente une augmentation de 1600%. De msme, la concentration des sucres, lies aux proteines, varie de 450% dans le cas du galactose. Les concentrations moyennes (FM) de sucres, lies aux proteines, sont: galactose, 395 & 235; hexosamine, 290 + 169; acide sialique, 151& 83; fucose, 160 h 108. Des s&cr&eurs de substances en rapport avec les groupes sanguins ont des concentrations de sucres, li6s aux protdines, plus ClevCs que les non-secreteurs, sauf en ce qui concerne l’acide sialique dont la concentration est identique dans les deux groupes. Le rapport acide sialique/fucose des Gcreteurs est de 0.64 & 0.12 et ne varie pas avec la vitesse de secretion. Le rapport pour des non-s&cr&eurs est de 1,40 L 0.35 et s’&ve avec l’augmentation de la vitesse de secretion. Zusammenfasstmg-Die Konzentrationen von Eiweiss und eiweissgebundenen Kohlenhydraten im menschlichen Maxillarspeichel hing von der Speichelfliessrate und nicht von der speziflschen Art des geschmacksreizes ab. “Unstimulierter” Speichel besass die hijchste Konzentration eiweissgebundener Kohlenhydrate. Niedrige Fliessraten waren mit den geringsten Konzentrationen an Eiweiss und eiweissgebundenen Kohlenhydraten kombiniert; wenn die Fliessrate anstieg, gab es ein adaequates Anwachsen der Konzentrationen dieser Substanzen. Die Konzentrationen der eiweissgebundenen Kohlenhydrate variierten im “unstimulierten” Speichel stark. Stimulierier Speichel war hinsichtlich der Zusammensetzung verhlltnismassig konstant. Die Eiweisskonzentration betrua im Mittel 122 i- 76 (S.D.) mgo/,, der Konzentrationswechsel vom niedrigsten zum-hiichsten beobachteten Wert st%e jedoch einen Anstieg urn 1600% dar. In &b&her Weise variierten die Konzentrationen der

HUMAN

SALIVARY

PROTEXE ANDGLYCOPROTEIN CONCENTRATIONS

449

eiweissgebundenen Koblenhydrate im Falle der Galaktose urn 4SO”/o. Die mittleren Konzentrationen (PM) betrugen fiir eiweissgebundene Kohlenhydrate: Galaktose: 395 -!-235; Hexosamin: 290 :‘I 169: Sialinsilure: 151 I- 83; Fukose: 160 2108. Ausscheider von Blutgruppensubstanzen besassen hiihere Konzentrationen an eiweissgebundenen Kohlenhydraten als Nichtausscheider mit Ausnahme der Sialinslurekonzentration, die bei beiden Gruppen iihnlich war. Dar Verhlltnis Sialinslure/ Fukose betrug fiir Ausscheider 0,64 -k O,t2 und variierte nicht mit der Fliessrate. Dass Verhlltnis fiir NichtalIsscheider betrug 1.40 :“:0.35 und stieg mit anwachsender Fliessrate an. REFERENCES ANREP,G. V. 1921. Therelation of the chorda tympani to the nitrogen metabolism of the submaxillary gland. J. Physiol. 54,319-331. ARAIU, Y. 1951. Nitrogenous substances in saliva. I. Protein and non-protein nitrogens. Jap. J. Physiol. 2,69-78, BOAS, N. F.

19.53. Method for the determination of hexosamines in tissues. J. biof. Chem. 204,5.53-563. BRAMKAMP, R. 0. 1936. The protein content of human parotid saliva. J. biol. Chem. 114,369-371. BUROEN,A. S. V. 1956. The secretion of potassium in saliva. J. Physiol. 132,20-39. BUROEN,A. S. V. and EMMELIN, N. G. 1961. Ph.vdologvofthe Salivary G/an& p. 182. Williams and Wilkins, Baltimore. CALDWELL, R. C. 1964. The Glycoproteins of Human S~tbmaxil1ar.vSecreriou. Ph. D. dissertation, University of Atabama. CALDWELL, R. C., and PIOMAN,W. 1964. Human submaxil1~ ~ycoproteins. J. dent. lies. 43,765. CALDWELL, R. C. and PIGMAN, W. 1965a. Disc electrophoresis of human saliva in polyacrylamide gel. Arch. Biochem. Biophys. 110,91-96. CALDWELL, R. C., and PIGMAN,W. 1965b. The carbohydrates of human submaxillary glycoproteins in secretors and non-secretors of blood group substances. Biochim. biophys. Acta. 101,X57-165. CHAUNCEY, H. H., and SHANNON, I. L. 1960.Parotid gland secretion rate as method for measuring response to gustatory stimuli in humans. Proc. Sot. exp. Biol. N. Y. 103,459-463. CLARK,E. P. 1943. Semimi~ro Qua~tj~a~jveOrganic Analysis, pp. 37-53. Academic Press, New York. DAWES, C. and JENKINS, G. N. 1964. The effects of different stimuli on the composition of saliva in man. J. PhysioI. 170,86-100. DEAKINS, M., CHEYNE, V. D., BIBBY, B. G. and VANKESTEREN, M. 1941. Significance of salivary analyses upon small groups of subjects. J. dent. Res. 20, 161-170. DJSCKE,2. 1955. Methods of Biochemical Analysis, Vol. II (Edited by GLICK,E.) p. 327. Interscience. New York. DISCHE,‘2.and SHETTLES, L. B. 1948. A specific colour reaction of methylpentoses and a spectrophotometric mi~omet~od for their dete~~nation. J. Mot. Chem. 175,595-603. FERGUSON, M. H., KRAHN,H. P. and HILDES,J. A. 1958. Parotid secretion of protein in man. fba. J. Biochem. Physiol. 36, 1001-1008.

KABAT,E. A. 1956. Blood Group Substances. Academic Press, New York. MANDEL, I. D., and ELLISON, S. A. 1963. The proteins of human parotid and submaxillary saliva. Ann. N. Y. Acad. Sci. 106,271-277. MANDEL,I. D., THOMPSON, R. and ELLISON, S. A. 1963. Carbohydrate components of submaxillary saliva. Intonations Association for Dental Research, Abstract 122. MANDEL,I. D., THOMPSON, R. and ELLISON,S. A. 1964. The carbohydrate com~nents of human submaxillary saliva. Arch. oral Biol. 9,601-609. NEWBRUN,E. 1962. Observations on the amylase content and flow rate of human safiva following gustatory stimulation. J. dent. Res. 41,459-465. PIGMAN, W., HAWKINS,W. L., BLAIR,M. G. and HOLLEY, H. L. 1958. Sialic acid in normal and arthritic human syaovial fluids. Arthritis Rheum. 1,151-166. SCHNEYER, L. H. 1955. Method for the collection of separate submaxillary and sublingual salivas in man. J. dent. Res. 34,257-261. SC-, L. H. 1956. Amylase content of separate salivary grand secretions of man. J. appf. Physioi. 9.453-455.

STAVRAKY, G. W. 1940. The partition of nitrogen in the submaxillary saliva evoked by chorda tympani stimulation in the cat. Amer. J. Phvsiol. 129.539-545. TJWENBALJM, B.,and KAR~HAN, M, 1939 Factors in saliva correlated with the occurrence of calculus. J. Amer. dent. Ass. 26,1965-1971. PLATEI OVERLY

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SALlVARY

PROTEIN

AND GLYCOPROTEIN

CONCENTRATIONS

FIG. 4. Effect of various stimuli on the pattern of salivary flow as observed on polygraph recordings of salivary drop rates. Numbers in parentheses indicate salivary Row rate.