STUDIES ON PEROXIDASE ACTIVITYAND

IODINE METABOLISM IN SALIVARY GLANDS

AKADEMISK AVHANDLINGSOM MED TILLSTAND AV

MEDICINSKA FAKULTETEN VID UMEÅ UNIVERSITET FÖR ERNÅENDE AV MEDICINE DOKTORSGRAD

OFFENTLIGEN FÖRSVARAS I SAL A, LUO-HUSET, UMEÂ UNIVERSITET, UMEÂ

TORSDAGEN DEN 16 MAJ 1974 KL 9.00

AV

ANDERS KUMLIENmed. lic.

UMEÅ 1974

UMEÀ UNIVERSITY MEDICAL DISSERTATIONS No 9, 1974FROM THE DEPARTMENTS OF OTO-RHINO-LARYNGOLOGY,

PHYSIOLOGICAL CHEMISTRY AND HISTOLOGY,UNIVERSITY OF UMEÀ, UMEÀ, SWEDEN.

STUDIES ON PEROXIDASE ACTIVITYAND

IODINE METABOLISM IN SALIVARY GLANDS

BY

ANDERS KUMLIENmed. lie.

UMEÂ 1974

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS No 9, 1974.EDITED BY THE MEDICAL FACULTY THROUGH THE DEAN,

PROFESSOR H. LINDERHOLM

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To my family

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This dissertation is a summary of the following publica­tions:

I. Iodine metabolism and peroxidase activity in salivary glands. Acta Endocrinologica 70 (1972) 239.

II. Cytochemical localization of peroxidase activity in the submandibular salivary gland ot the hamster. Histochemie 22 (1970) 294. (In collaboration with G.D. Bloomand B. Carlsöö).

III. A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man. Histochemie 26 (1971) 80. (In collabora­tion with G.D. Bloom and B. Carlsöö).

IV. Iodide accumulation and peroxidase activity in the sub­mandibular salivary gland of the normal and castrated male hamster. Archives of Oral Biology (In press). (In collabora­tion with G.D. Bloom and B. Carlsöö.)

V. A method for radiosialometry: Studies on salivary secre­tion, iodide-concentrating ability, and iodide clearance of the parotid and submandibular glands in man. Archives of Oral Biology (In press). (In collaboration with A. Wiberg.)

In the following discussion the papers will be referred to by the Roman numerals I—V.

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Introduction

In certain sialoses and inflammatory processes affecting the salivary glands, the patient is troubled by a sensation of dryness in the mouth and throat. This has been attributed to degenera­tive changes in the major salivary glands and in the small sali­vary glands in the mucous membrane of the oral cavity. (Chisholm and Mason, 1969; Whaley et al., 1968; Whaley and Buchanan, 1971.) The condition is referred to as xerostomia (Bertram, 1967). In a milder form, this problem is frequently encountered in the clinic, but it is seldom possible by means of sialometry or sialography to demonstrate changes in the salivary glands in this group of patients. On the other hand, inflamma­tory and neoplastic changes in the salivary glands and sialoses of various types are regarded as fairly rare conditions.

Enlargement of the salivary glands has almost been regarded as synonymous with pathology of these glands (Rauch, 1959; Diamant, 1960; Borsanyi, 1962; Hall, 1969). Acute or chronic inflammatory conditions of the salivary glands seldom consti­tute a diagnostic problem. Since puncture biopsy has come into widespread clinical use, this is also true of neoplastic changes. Sialoses, on the other hand, often present a problem in differen­tial diagnosis. Rauch in 1959 presented an elaborate classifica­tion of the causes of salivary gland enlargement, and Diamant (1960) proposed a division into four groups for clinical pur­poses:

1) Rheumatoid or collagen type2) Sarcoidosis3) Asymptomatic type with or without hormonal insufficiency4) Chronic recurrent parotitis.

Group 4 seldom poses a problem in differential diagnosis, but the other groups may, especially in the early stages. The asymptomatic type may be associated with hormonal imbalance or with conditions such as diabetes mellitus, diseases of the thyroid, puberty and the menopause (Borsanyi, 1962; Anger- vall, Dotevall and Westling, 1963, Diamant, 1966; Davidson, Leibel and Berris, 1969; Diamant and Enfors, 1970; Miinzel, 1971) or with other disease states such as cirrhosis of the liver,

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malnutrition and gout or alcoholism and intoxications (Roth- bell and Duggan, 1957; Borsanyi and Blanchard, 1961; Borsa- nyi, 1963; Goobar and Goobar, 1965; Seifert, 1965). Salivary gland enlargement may also occur as an isolated phenomenon (Borsanyi, 1962; Diamant and Enfors, 1970; Miinzel, 1971). Greenberg et al. (1964) found an enlargement of the parotid glands in 6 % of 388 patients with sarcoidosis. It is reasonable to assume that salivary gland pathology may be present without affecting the size of the gland. Bhoola et al. (1969) demonstra­ted a decrease in saliva production and in the enzyme content (amylase and kallikrein) of the saliva in certain patients with sarcoidosis in whom there were no other clinical signs of sali­vary gland involvement.

The methods currently available for evaluating the functional state of the salivary glands are, in part, qualitative: anamnesis, physical examination, sialography and cytological examination of biopsy material; in part, quantitative: sialometry and deter­mination of the amount of technetium taken up by the salivary glands (Hall, 1969; Whaley and Buchanan, 1971; Brands, 1972). There are large normal variations in the values obtained by these quantitative methods, but correlation of the sialometric findings with data on the size of the gland obtained by sialography increases the possibility of correctly estimating the functional capacity of the salivary glands (Ericson, 1971). Serological tests for autoimmune factors and serum electrophoresis are also of value. (Blatt, 1965; Whaley and Buchanan, 1971.)

In addition to diagnosis, it is important to determine the extent of any damage present. This, however, can seldom be done by means of the methods mentioned above.

Determination of concentration and clearance of various sub­stances and analyses of enzyme content would probably in­crease the possibility of diagnosing a pathological condition in an early stage and determining its nature and extent. Early diagnosis is important, partly in order to elucidate the patho­genesis of a disease state and partly to facilitate therapy. However, no tests of concentration and clearance for evaluation of the functional state of the salivary glands seem to have been published.

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The saliva normally contains a large number of electrolytes and proteins (Thaysen et al., 1954; Rauch, 1959; Brands, 1972; Schneyer et al., 1972) which are metabolized in the salivary glands. Of the electrolytes in the saliva — mainly potassium, calcium, sodium, phosphate, sulfate, bicarbonate, chloride, thiocyanate and iodide — I chose to study the metabolism of the iodide ion in the salivary glands, since this ion is concen­trated in the tubular system of the glands in the hamster (Cohen, Myant and Logothetopoulos, 1955; McGee, Mason and Duguid, 1967; Rogers and Brown-Grant, 1971) and in the dog (Burgen and Seeman, 1957; Burgen, Terroux and Gonder, 1959), and if there is a similar capacity to concentrate iodide in man, it would probably be differently affected by different disease states, depending on which type of cell in the gland is damaged first or most extensively. In addition, iodide isotopes have long been used in medical diagnostic procedures and equipment for iodide determination is to be found in most larger hospitals.

Of the proteins, I chose to study the enzyme peroxidase, since it is known that iodide and peroxidase often interact in metabolic processes, e.g. in the synthesis of iodoproteins in the thyroid gland (Ljunggren, 1957; Saunders and Stark, 1958) or when the iodide ion potentiates the antibacterial effect of peroxidase (Klebanoff, 1968 a, b).

Iodide is concentrated and metabolized by a number of organs such as the thyroid gland, the salivary glands, the lacta- ting breast, the intestine, the ovum and the placenta (Brown- Grant, 1961). The ability of human salivary glands to take up iodide from the serum and to concentrate it and excrete it in the saliva was probably recognized by Claude Bernard as early as 1856 (Burgen and Seeman, 1957). In the thyroid, iodide is taken up from the serum and bound to proteins, but in salivary glands, the mammary glands and in the intestine, iodide is taken up and concentrated and then excreted in saliva, milk or serum; in these tissues iodoproteins are probably not synthesized in vivo (Taurog, et al., 1959; Myant, 1960; Brown-Grant, 1961).

It is further known that, like the lactating breast, certain salivary glands contain the enzyme lacto-peroxidase (Alexander, 1959, Morrison and Allen, 1963; Thomson and Morell, 1967).

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The antibacterial effect of this enzyme is enhanced by iodide (Klebanoff, 1968 a, b), but not all salivary glands have the ability to concentrate iodide (Cohen and Myant, 1959).

The question whether there is a positive correlation between the presence of peroxidase and the ability of various salivary glands to concentrate iodide was unanswered, and motivated such a study. The cellular localization of peroxidase in salivary glands was also unknown.

Present investigation

This investigation was aimed at studying1. the correlation between the presence of peroxidase activity

and iodide-concentrating capacity in the parotid, submandi­bular and sublingual glands of man and various animals;

2. iodide metabolism in salivary glands of various types;3. the cellular localization of salivary gland peroxidase;4. the cellular localization of the ability to accumulate iodide

and its dependence on hormones (effects of castration);5. the ability of human parotid and submandibular glands to

concentrate iodide to the saliva, the normal variations of this concentrating ability and of iodide clearance and the mutual interdependence of various types of salivary glands.

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Correlation between the presence of peroxidase activity and the iodide-concentrating ability of salivary glands of

different types from man and different animal speciesCertain salivary glands are able to concentrate iodide while

others lack this ability (Cohen and Myant, 1959). Peroxidase activity has been demonstrated in certain salivary glands and in the saliva (Alexander, 1959; Morison and Allen, 1963; Thomson and Morell, 1967; Mosimann and Sumner, 1951).

The peroxidase activity in homogenates of the parotid, sub­mandibular and sublingual glands of man, dog, cat, rabbit, guinea pig, hamster, rat and mouse (24 types of glands) was determined according to the guaiacol method (Chance and Maehly, 1955) (I.) Glands in which the quotient

iodide/Kmm>1 (accumulation) or the quotient

iodide/Ad semm >1 (concentration) were defined as glands withthe ability to concentrate iodide. According to this definition, three of the 24 different salivary glands (parotid and submandi­bular gland ot the cat, which contain peroxidase activity, and the submandibular gland of the dog, which lacks peroxidase activity) were not classifiable, since the quotients were close to 1. Thus the ability of these glands to accumulate or concen­trate iodide must be regarded as slight or lacking. In 19 of the 21 investigated glands that could be classified, the ability to concentrate iodide and peroxidase activity were both present in 12 glands and both absent in 7. The submandibular gland of the mouse and the parotid gland of the rabbit, however, were able to concentrate iodide, but had no detectable peroxidase acti­vity, according to the assay used. Thus is appeared that iodide can be concentrated from serum to saliva in the absence of peroxidase.

In summary, peroxidase activity and the ability to concen­trate iodide were both present in 12 glands and both absent in 8. Peroxidase activity but not the ability to concentrate iodide was present in 2 glands, and finally 2 glands were capable to concentrate iodide but had no peroxidase content.

Then we can make a chi2-test of the null hypothesis that peroxidase activity and ability to concentrate iodide are inde­pendent. The critical chi2 value at the 5 % level is 3.8. As the

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observed value exceeds the critical value, the null hypothesis must be rejected. Thus there is a dependence between peroxi­dase activity and ability to concentrate iodide.

No correlation could be detected between the functional pro­perties ”iodide-concentrating ability” or ”peroxidase activity” and the histological classification of the glands as serous, mucous or mixed type. For a detailed histochemical investiga­tion, the submandibular glands of man and various animals were chosen, as the relatively high fat content of the parotid gland gives rise to certain technical problems. Peroxidase activity could only be demonstrated in serous cells in acini or in excre­tory cells in the tubular system in the submandibular glands of man, hamster and guinea pig (II, III). Carlsöö (1971) and Bloom and Carlsöö, (1974) have demonstrated abundant peroxidase activity in the seromucous cells in acini of the bovine submandi­bular gland, whereas the mucous cells are completely lacking in such activity.

Iodide metabolism in the salivary glandsSalivary glands with peroxidase activity and the ability to

concentrate iodide contain the necessary conponents for syn­thesis of iodoproteins, sometimes in the same type of cell (i. e. in the GCT cells in the hamster submandibular gland). Iodo- amino acids and iodoproteins have been synthesized in vitro in the presence of iodide, salivary gland homogenate or pieces of salivary gland, and a system capable of generating H2 02 (Kes- ton, 1944; Alexander, 1959; Hati and Datta, 1967). These reactions, however, were probably due to nonspecific oxidation. Current opinion seems to be that iodide during its metabolism in salivary glands in vivo and in saliva remains in inorganic form, and is not bound to proteins (Taurog et al., 1959; Myant, 1960; Brown-Grant, 1961). The results of in vivo studies have changed as the available methods have improved. Thus Honour et al. (1952) found that an appreciable part of the 1311 activity in human saliva could be precipitated with trichloroacetic acid. Logothetopoulos and Myant (1956) chromatographed a homo­genate of hamster salivary glands extirpated after the injection of 1311 and found some activity in the position for iodopro-

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teins but 90 % of the activity in the position for iodide. Ger- baulet and Fitting (1956) found on paper electrophoresis that 10—15% of the 1311 activity in the saliva of a hyperthyroid patient was in inorganic form, and Cohen and Myant (1959) found on chromatography of homogenate of human parotid and submandibular glands that about 5 % of the activity was organically bound. Freinkel and Ingbar (1953) chromato­graphed saliva from man, while Ruegamer (1955) used saliva from dogs after injection of 1311. They found only 1311 iodide in the saliva. Taurog et al. (1959) found inorganic iodide on chromatograms of mouse submandibular gland homogenate only during the first hours after 1311 injection, but after 24 hours the same authors found between 1.4 and 6.3 % of the activity in the position for iodoproteins.

Howewer, only a limited number of the salivary glands that accumulate iodide have been studied in this respect, and as is apparent from the above, the methods used have varied widely. In addition, studies have not previously been conducted on sali­vary glands that lack the ability to accumulate iodide but which are able to concentrate iodide to the saliva (guinea pig) or on glands that lack the ability to concentrate iodide. Although iodide is probably only turned over as iodide in salivary gland metabolism, all the possible combinations in this respect have not been investigated.

Taurog et al. (1959) used methylthiouracil in the homo­genate to prevent formation of iodoproteins in vitro, a method which I have also used. A chromatographic analysis with respect to labelled iodoproteins, 1311-iodinated amino acids and 1 311" was therefore performed on various salivary gland homogenates, saliva, thyroid homogenate, serum and standard solutions.

The method of preparing homogenates and the chromato­graphic procedures used for saliva and thyroid gland homoge­nates are given in (1). For chromatography of human saliva (for collection of saliva samples, see V) 0.5 ml of saliva was added to 0.5 ml of buffer containing 1 mM methylthiouracil. 120 pi amounts of this mixture were chromatographed on Whatman No 1 paper according to Taurog et al., 1950, Taurog and Chaikoff, 1957. Figure 1 shows chromatograms of standard solutions.

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c.p.m

Fig. 1. Chromatograms of standard solutions (A and B) in collidin-HO-ammonia atmosphere to the left and in butanol-ethanol-2N ammonia to the right. (O = origin; SF = solvent front; c.p.m. = counts per min; Calculated Revalues are given.)

A) 1311'as Nal.

B) 131F labelled human serum albumin.

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300-

200-

100 —

c.pm

Fig. 2. Chromatograms of salivary gland homogenates one hour after intraperitoneal injection of 1 *ras Nal. Chromatograms in collidimi^ O-ammonia atmosphere to the left and in butanol-ethanol-2N ammonia to the right. (0 = origin; SF = solvent front; c.p.m. = counts per min; Calculated Revalues are given.) In C and D the line c.p.m. = 4 indicates the highest level to which the background activity fluctuated.

A) A salivary gland which accumulates iodide and concentrates iodide to the saliva but contains no peroxidase. (Mouse submandibular gland.)

B) A salivary gland which accumulates iodide and concentrates iodide to the saliva and contains a peroxidase. (Cat sublingual gland.)

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C) A salivary gland which concentrates iodide to the saliva but has no ability to accumulate iodide. The salivary gland contains a peroxidase. (Guinea-pig submandi­bular gland.)

D) A salivary gland with no ability to concentrate or to accumulate iodide. The salivary gland contains no peroxidase. (Rat submandibular gland.)

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c.pxm

Rf*0.78 SF

Fig. 3. Chromatograms of thyroid gland homogenates and serum in ^colli- din-FLO-ammonia atmosphere one hour after intraperitoneal injection of 1 11 as Nal. (D = origin; SF = solvent front; c.p.m. = counts per min. Calculated Revalues are given).

A) Thyroid gland of hamster.

B) Serum from rat.

Mouse, rat, hamster, guinea pig, rabbit and cat received Na1311 intraperitoneally and were killed after 1 or 24 hours. The parotid, submandibular and sublingual glands were homo­genized and 20 jul amounts of homogenate were chromato­graphed in two systems. The chromatograms were analyzed for 1311 (Fig. 2).

Thyroid gland specimens and serum were chromatographed under the same conditions (Fig. 3).

With homogenates from thyroid glands from animals given 13 11 in the same way, most recovered activity appeared at the position for iodoproteins, some activity was recovered in the position for iodide, and a small amount of radioactivity at Rf<I" probably represented iodo-amino acids. M ethylthiouracil was also added in these experiments.

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ïl10 15 SF 20

Fig. 4. Chromatograms of human parotid^A) and submandibular (B) saliva 7.5-9.0 min after intravenous injection of 1 as Nal. Chromatograms in colli- din-H2O-ammonia atmosphere to the left and in butanol-ethanol-2N ammonia to the right. (O = origin; SF = solvent front; c.p.m. = counts per min; Calculated Revalues are given.) The line c.p.m. = 4 indicates the highest level to which the background activity fluctuated.

The same analyses were performed on human parotid and submandibular saliva collected 7.5—9.0 min after intravenous injection of Na1 3 11 (Fig. 4).

Iodoproteins or iodo-amino acids were not demonstrable in salivary gland homogenates, serum or saliva in either of the two chromatographic systems, except in the homogenates of the parotid and submandibular glands of the hamster, where very small amounts (0.5 % of the recovered activity) were found bound to proteins (I). This activity was interpreted as a techni­cal artefact, probably due to nonspecific iodination of proteins as a result of the high concentrations of iodide and peroxidase in the homogenates.

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The cellular localization of salivary gland peroxidase

Salivary gland peroxidase is readily soluble, has a moleculai weight of about 78 000 (Morrison et al., 1966), and is immuno- logically and spectrophotometrically identical to milk peroxi­dase (lactoperoxidase) (Morrison and Allen, 1963; Morrison et ah, 1965). In a study involving differential centrifugation of homogenate from the submandibular gland of goat, the enzyme was traced to a microsomal fraction (Hati et al., 1968), but Thomson and Morell (1967) found the enzyme in the super­natant of a homogenate from the submandibular gland of the ox.

Submandibular glands from man, dog, rabbit, guinea pig, hamster and rat were examined histochemically for peroxidase activity. (Methods in II, III and IV). These six different types of submandibular gland were chosen on the basis of their different peroxidase content, abilities to accumulate and concentrate iodide and their histological structure (Table I).

Table I

Species Iodide- Iodide-accumulating concentrating ability ability

Serous (S), Mucous (M) or Serorrucous (SM)

P.O

Man + + SM 2.9Dog _ _ M 0.0Rabbit — — SM 0.0Guinea pig — + S 3.3Hamster + + SM 10.0Rat — — S 0.0Iodide accumulating and concentrating ability according to Cohen and Myant (1959), histological classification of the acini according to Shackleford and Klapper (1962) and Shackleford and Wilborn (1967), and peroxidase activity according to Kumlien (1972) in the submandibular salivary glands. P.O. = Mean peroxidase activity: ^A470mjU/min/ml homogenate.

Peroxidase activity could be demonstrated in the granules of the serous cells in the acini in four of seven human submandi­bular glands, in the cells of the granular convoluted tubules

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(GCT cells) of the submandibular gland of the hamster and in the acinar cells and the secretory tubular cells in the submandi­bular gland of guinea pig. Small granules in the cytoplasm of collecting duct cells were peroxidase-positive in the submandi­bular gland of the dog. These granules appear to be lysosomes (Carlsöö, 1973). With this exception, peroxidase activity was not demonstrable in the submandibular glands of dog, rabbit or rat (II and III).

The cellular localization of iodide-accumulating ability and the effect of castration

In 1956 Logothetopoulos and Myant, using autoradiographic technique, found the iodide-concentrating cells to be localized to the tubules in the parotid glands of mouse and hamster and to the granular convoluted tubular cells (GCT cells) in the sub­mandibular glands. Tilmann (1967), also using autoradiographic technique, found that iodide is accumulated in the acini as well as in the tubules of the guinea pig submandibular gland. The iodide-concentrating cells in the parotid gland of the dog are located in the proximal parts of the distal tubules (Burgen et al., 1959).

The salivary glands are affected by the state of hormonal balance. When the latter is disturbed, hormonal sialoses may develop (Borsanyi, 1962; Davidson, Leibel and Berris, 1969; Miinzel, 1971). The salivary glands of the mouse are under hormonal control. For example, the histological appearance of the granular convoluted tubules (GCT) is altered in castrated male mice (Brown-Grant and Taylor, 1963) and the ability to accumulate iodide is also altered (Llach and Tramezzani, 1962; Brown-Grant and Taylor, 1963). Iodide-accumulating ability and peroxidase activity in the hamster submandibular gland were therefore also studied in castrated male hamsters (IV).

Iodide is a readily diffusible ion. There are usually high con­centration gradients across the cell membranes, which can easily lead to diffusion artefacts when autoradiographic techniques are used. On the other hand, it is difficult to localize the iodide­concentrating cells by any other technique.

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In this study (IV) several techniques were used simulta­neously on the same glandular specimen. Peroxidase activity was determined histochemically by means of a diaminobenzi- dine (DAB)-H2 02 method (IV) and quantitatively in a homo­genate of the same tissue with the guaiacol method (I). Accumulation of iodide in the gland was studied in part by a modified autoradiographic method for readily diffusible substances and in part by quantitative determination of the 12 51 activities in salivary gland homogenate and serum. A special box was constructed for use with the modified auto­radiographic technique (Fig. 5).

TISSUE SECTIONS

•ILEORD G 5 GLASS

RUBBER RING

SILICONIZED GLASS

Fig. 5. Light-tight box for autoradiography of tissue sections. The sections are placed on a siliconized glass slide and face an emulsion (Ilford G5)-coated slide. Good contact between section and emulsion is ensured by bulldog clips which hold top and bottom of the box together.

Peroxidase activity and iodide-accumulating ability were demonstrated in the cells of the granular convoluted tubules. Castration of the male hamster had no discernible effect on cellular morphology, localization and amount of peroxidase activity or on the ability of the submandibular gland to accumulate iodide (IV).

The capacity of human parotid and submandibular glands to produce saliva and to concentrate iodide to the saliva

under conditions of submaximal stimulation with citric acid

The ability of the parotid and submandibular glands to pro­duce saliva and to excrete iodide to the saliva was studied by simultaneous registration from the four large salivary glands in 27 men under oral stimulation with 6 % citric acid (V) (Fig. 6). This type of stimulation of saliva production was chosen because it is simple to perform and does not require intravenous

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INJECTION 131 I BLOOD SAMPLE A B C

■--------1-------- .-------- 1--------- 1-------- .-------- •--------- 1-------- •-------- 1-------- 1--------- .-------- 1---------------------------------------------> TIME0 1' 2' 3' 4' 5' 6' 7' 8' 9' 10' 11' 12'

SALIVA SAMPLE I II III

CITRIC ACID STIMULATION

131Fig. 6. Procedure of study. 1-2/iCi I iodide was slowly injected during the first minute. Citric acid (6 per cent) stimulation was applied 4 x per min during the time interval 4 min 30 sec to 10 min 30 sec. Saliva samples were colleted from both parotid and submandibular glands during three collection periods.

Period I : from 6 min to 7 min 30 sec,Period II: from 7 min 30 sec to 9 min,Period III: from 9 min to 10 min 30 sec.

Blood samples were collected during the time intervals 6 min 30 sec to 7 min (A), 8 min 45 sec to 9 min 15 sec (B) and 11 min 15 sec to 11 min 45 sec (C).

Ri ght + left parotidsecretionrate.

(m l/min)

to salivationIncreasing stimulation

Fig. 7. Mean values for the sum of the right and left human parotid secretion rates (ml/min under resting conditions (R) and in response to 1,6 and 10 per cent (w/v) citric acid. Numerical values from Ericson, 1969, 1971.) N = 92 (61 <j>; 31 o).

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injection of pharmacological agents which have effects on other organs, such as, for example, acetyl-beta-methylcholineiodide (Betacholyl®, Vitrum, Stockholm) or synstigmine methylsul- fate Prostigmin®, Roche, Basel, Switzerland). Further, the pro­cedure of stimulation and the secretory response are highly reproducible (Ericson, 1968). Figure 7 shows the rate of sali­vary secretion from the human parotid gland in the resting state under oral stimulation with citric acid of increasing concentra­tions, according to Ericson (1969, 1971). At high concentra­tions of citric acid (6—10 %) there is less variation of the secre­tory response than when low concentrations are used (resting conditions — 1 %). At low concentrations of citric acid, the intensity of stimulation may vary due to dilution by varying amounts of saliva in the oral cavities of different individuals. For these reasons I used a high concentration of citric acid to stimulate salivary secretion. Howewer, the long period of sti­mulation to be used (6 min) precluded the use of 10 % citric acid, since there might well be a risk of damage to the epithelium of the tongue. Stimulation was therefore carried out with 6 % citric acid and was judged to be submaximal.

The ability of the parotid and submandibular glands to secrete saliva and to concentrate iodide to the saliva under conditions of submaximal stimulation showed a great deal of variation (Figs. 8 — 11). From the values obtained, the iodide clearance was calculated for each gland. In the histograms of iodide clearance, most of the measured values are near the lower limit of normal (2.5 percentile) (Figs. 12 and 13). The inter-in- dividual and intra-individual variations in the ability of the glands to produce saliva, to concentrate iodide from serum to the saliva and to clear iodide were determined non-parametri- cally with the 2.5 and 97.5 percentiles as limits (V).

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LOzo

erLULOCDo

erLUCD

Z)Z

12n10

9

t5

43

2

1

PAROTID GLAND SECRETION

0 30 045SECRETION ML/MIN

P2 5 P50 P975

Fig. 8. Distribution of parotid secretion in response to 6 per cent citric acid stimulation for the 54 parotid glands of 27 normal male students. The median secretion (P50) = 1-07 ml/min; 0*2 5 = 0-47 ml/min; Pgj 5 = 1.70 ml/min.)

tnc

>erLULOCDc

erLUCD2

1ZH1Û9

8

6

5

V32

SUBMANDIBULAR GLAND SECRETION

1

0 35 065

25

095 110 1 25

50

155 185SECRETION* ML/MIN

P97 5

Fig. 9. Distribution of submandibular secretion in response to 6 per cent citric acid stimulation in 54 submandibular glands. The median secretion (P 0) = 0-98 ml/min; (P2 5 = 0.45 ml/min; Pgj ^ = 1.78 ml/min.)

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IODIDE CONCENTRATING ABILITY IN HUMAN PAROTID GLANDS

Fig. 10. Distribution of iodide concentration ratios (Rp) in saliva from glands after stimulation with 6 per cent citric acid.

131- I-activity/ml saliva(Rp) = rît---------- —------- *I-activity/ml serum

The median (Rp)-value (P^q) = 15.1; (?2 5 = ^*^’^97.5 = 34.6.)

CONCENTRATIONRATIO

54 parotid

14

12-

10

CO

o 8<>or 6-LU 'COCQ° 4LLow 2CO2:3z 0

IODIDE CONCENTRATING ABILITY IN HUMAN SUBMANDIBULAR GLANDS

28

_____ CONCENTRATIONRAT!0(*S/f)

r25 r50 r97.5

Fig. 11. Distribution of iodide concentration ratios (R<^) in saliva from 54 sub­mandibular glands after stimulation with 6 per cent citric acid.

- 131 I-activity/ml saliva

(Rsm) = ;I-activity/ml serum

The median(Rgj^)-value(P50) = 15.3; (?2 5 = 8.0; P97 ^ = 21.7.)

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ODI DE CLEARANCE IN HUMAN PAROTID GLANDS

25

IODIDE CLEARANCE ML/M IN

Fig. 12. Distribution of salivary iodide clearance values for 54 parotid glands submaximally stimulated. The median value (P^q) = 14.1 ml/min; (?2 5 = 7.5 ml/min; P^y ^ = 32.8 ml/min.)

IODIDE CLEARANCE IN HUMAN SUBMANDIBULAR GLANDS

Fig. 13. Distribution of salivary iodide clearance values for 54 submandibular glands under submaximal stimulation with 6 per cent citric acid. The median value (P<-q = 14.9 ml/min; (P2 5 = 5.4 ml/min; P^y ^ =31.6 ml/min.)

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There was a positive correlation between saliva production by the parotid gland and by the submandibular gland, and between the iodide clearances by the two types of glands under submaxi­mal stimulation. No correlation between the iodide-concentra­ting ability of the parotid gland and that of the submandibular gland could be established with certainty under the same type of stimulation. The inter-individual variations were considerably greater than the intra-individual with respect to all three para­meters: salivary secretion, iodide-concentrating ability and iodide clearance. (V)

General discussion

Peroxidase is present in certain salivary glands, but not in all (I). A dependence between the occurrence of peroxidase acti­vity and the ability of salivary glands to concentrate iodide suggests that both properties are involved in a common func­tion. Of 24 different types of salivary glands studied, peroxidase and iodide-concentrating ability were both present in 12 and both lacking in 7. In three glands the concentrating ability was difficult to evaluate and was judged to be either extremely weak or absent. In two of these glands (the parotid and submandi­bular glands of the cat) there was abundant perioxidase activity, and in the third no peroxidase activity was detectable on quan­titative chemical analysis. A further two glands (parotid gland of rabbit and submandibular gland of mouse) were able to con­centrate iodide but had no detectable peroxidase activity. Thus it is apparent that iodide can be concentrated, transported in- tracellularly and secreted in the absence of the enzyme peroxi­dase.

There seems to be no correlation between the occurrence of salivary gland peroxidase or the ability to concentrate iodide, on the one hand, and diet or zoological classification of the animal, on the other. Neither is there any common embryolo- gical origin for the tissues that show iodide-concentrating ability (salivary glands, stomach, intestine, mammary glands and thyroid gland) in the adult human or adult animal.

25

The lack of protein-bound iodine in salivary gland homo­genates suggests that salivary gland peroxidase is involved in some function other than the synthesis of iodoproteins.

In histochemical studies of the cellular localization of peroxi­dase in salivary glands (II and III), peroxidase-positive granules were demonstrated only in excretory serous cells in acini and in granulated cells of excretory tubules. Peroxidase activity was also demonstrated in guinea pig saliva. Since iodide that is taken up from serum by salivary glands is rapidly concentrated and excreted to the saliva in unaltered form, it is probable that iodide has its metabolic function in saliva. Salivary gland peroxi­dase is secreted into the saliva together with amylase (Carlsöö and Bloom, 1972; Carlsöö et al., 1972, 1974; Bloom et al., 1974) Catecholamines are more potent inducers of enzyme sec­retion than are cholinergic agents (Hall, 1969; Carlsöö et al., 1972). The salivary gland peroxidase, like amylase, probably plays its metabolic role in the saliva.

Iodide ions are electron donors to the peroxide-peroxidase complex, and in this way iodide is able to function as a cofactor of peroxidase in peroxidative reactions (Saunders and Stark, 1958; Hati et al., 1968). The antibacterial (Klebanoff and Luebke, 1965), antimycotic (Lehrer, 1969) and viricidal (Belding and Klebanoff, 1970) effects of peroxidase are poten­tiated by halide ions, especially by iodide (Klebanoff, 1968 a, b; Iwamoto, Tsunemitsu and Okuda, 1972). It is therefore possible, and even probable, that peroxidase and iodide ions in the saliva constitute an antibacterial system in the ducts of the salivary glands and in the oral cavity.

Castration of male hamsters had no discernible effect on the histology, peroxidase activity or iodide-accumulating ability of the submandibular gland (IV). However, hormonal dependence of the mouse submandibular gland has previously been demon­strated (Lacassagne, 1940; Llach and Tramezzani, 1962; Brown-Grant and Taylor, 1963; Ferguson and Stephen, 1972), and salivary gland enlargement is a well-known phenomenon in humans with hormonal disturbances (Borsanyi, 1962; Angervall, Dotevall and Westling, 1963; Diamant, 1966; Davidson, Leibel and Berris, 1969; Diamant and Enfors, 1970;Miinzel, 1971).

26

Normal values obtained for saliva production, iodide-concen­trating ability and iodide clearance by the parotid and subman­dibular glands in 27 men are presented (V).

1 min 30 sec. after stimulation to salivation was started, saliva samples were collected during a period of 4 min 30 sec. During this period, secretion volumes, calculated values for iodide concentration and clearance remained constant. These findings are in accord with observations made by Diamant, Diamant and Holmstedt in 1957 (secretion volumes) and Stephen, Harden and Mason in 1971 (iodide concentration).

The salivary iodide concentration is inversely related to flow rate: an increasing rate of salivary flow leads to a decreasing salivary iodide concentration (Burgen and Terroux, 1962; Mason, Harden and Alexander, 1966). However, at parotid sali­vary flow rates greater than 1 ml/min the saliva/serum ratio for iodide remains constant (Harden and Alexander, 1967). Accor­ding to these findings, a submaximal stimulation was used to secure a high saliva secretion rate and a constant saliva/serum ratio for iodide. However, under these conditions of submaxi­mal stimulation with 6 per cent citric acid the saliva/serum iodide concentration ratio remained constant only for the human submandibular gland, whereas in the parotid gland the iodide concentration ratio fell as the flow rate increased (V). No explanation of this difference between the glands has been found.

The inter-individual variation was considerably greater than the intra-individual difference with respect to all three para­meters. The small side differences observed for normal patients can thus be valuable for estimation of asymmetric functional disorders. The clearance values did not show a normal distribu­tion, most of the observations being in the low normal range.

Stephen et al. (1973) found a positive correlation between the saliva/plasma concentration ratios for iodide in the human parotid and submandibular salivary glands. However, this obser­vation was made at fixed salivary secretion rates (0.5 and 1.0 ml/min). In our investigation, no correlation could be establis-

27

hed between salivary iodide concentration in the parotid and submandibular glands. However, in our investigation the stimu­lation to salivation was invariable and submaximal.

In the salivary glands, technetium is mainly metabolized by the same enzyme system as iodide (Alexander, Harden and Shimmins, 1968; Harden et al., 1968 a, b).

Salivary gland scanning (”radiosialography” according to Gates, 1972) or measuring the accumulation of 9 9m Tc-pertechnetate in the salivary gland (”radiosialometry” according to Lind and Söderborg, 1971) is mainly performed in patients with two types of salivary gland diseases — partly to detect ”hot areas” indicating the presence of Whartin’s tumors (Stebner et al., 1968; Gates, 1972) partly to search for a decrea­sed accumulation of the isotope indicating a decreased function of the salivary glands (Stephen et al., 1971; Schall and Di Chiro, 1972;Lafayeetal., 1973).

The tubular cell component is prominent in Whartin’s tumor (Stebner et al., 1968). The high 99mTc uptake in the tumor indicates that, as in most animals, 99mTc (and iodide) is accu­mulated in the human parotid duct cells.

28

General summary

1. A dependence between the presence of iodide-concentrating ability and peroxidase activity in the salivary glands of a number of animal species has been demonstrated.

2. Iodide administered is recovered as iodide in the salivary glands and in the saliva, regardless of whether these have peroxidase activity and/or the ability to concentrate iodide.

3. Salivary gland peroxidase is localized in granules. Peroxidase­positive granules have been demonstrated in the serous cells in the acini of the human submandibular gland, in the granular convoluted tubular cells of the hamster submandi­bular gland and in both the acinar and the tubular cells of the guinea pig submandibular gland as well as in guinea pig saliva. Peroxidase activity was not demonstrable in the submandi­bular glands of dog, rabbit or rat.

4. Peroxidase activity and iodide-concentrating ability are locali­zed to the same cells of the male hamster submandibular gland, and are not affected by castration. A simplified auto­radiographic method for detection of readily diffusible sub­stances has been developed.

5. The abilities of the human parotid and submandibular glands to secrete saliva and to concentrate iodide to the saliva have been studied. Inter- and intra-individual variations in these abilities have been determined in a material consisting of 27 men 19—26 years of age, average age 22 years.

6. It is proposed that salivary gland peroxidase and iodide con­stitute a system of defense against infection in the saliva and in the oral cavity.

29

Acknowledgemen ts

The investigation reported here was carried out at the Depart­ments of Physiological Chemistry, Histology and Oto-rhino- laryngology, University of Umeå, Umeå, Sweden.

Many thanks are due to all whose help has made the inves­tigation possible:

to Professor K-G. Paul, who suggested the subject of the inves­tigation. His interest, advice, criticism and help was of inestimable value.

to Professor H. Diamant for the numerous means by which he facilitated my investigation, his stimulating criticism and help in the course of work.

to Professor G. Bloom for his interest and intimate collabor­ation, help in the course of work and for guidance with the histologic and autoradiographic analysis.

to J. Granåsen, B.Sc., Ass. Professor Agne Wiberg and Ass. Professor T. Stigbrand for their interest and intimate collabora­tion as regards the statistical problems of this work.

to Professor J. Wersäll for support and encouragement.

to L. Jonsson, Miss M.Borg,Miss K. Karlsson and P.-I Olsson for exellent technical assistance during various phases of this work.

to Miss B. Steele for help with translation.

to my wife for having patience and giving me support at all times.

30

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