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Anesth Analg 2000;91:467-472
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

The Effects of Glycopyrrolate on Oral Mucous Host Defenses in Healthy Volunteers

Merja T. Lähteenmäki, MD*, Matti S. Salo, MD, PhD*, Jorma O. Tenovuo, DOdont, PhD{dagger}, Antti V. Helminen, MD{ddagger}, Pekka J. Vilja, PhD, and Risto K. Huupponen, MD, PhD{ddagger}

*Department of Anesthesiology and Intensive Care, {dagger}Institute of Dentistry, and {ddagger}Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku; and Department of Clinical Science, University of Tampere, Tampere, Finland

Address correspondence and reprint requests to Matti Salo, MD, PhD, Department of Anesthesiology and Intensive Care, University of Turku, FIN-20520 Turku, Finland. Address e-mail to matti.salo{at}tyks.fi


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We studied the effects of glycopyrrolate on oral mucous host defenses. Single IV doses of glycopyrrolate (4 µg/kg) or placebo were administered to 12 healthy volunteers in a randomized, double-blinded, cross-over study. Salivary flow rates and the concentrations/activities of total protein, amylase, and nonimmunologic (lysozyme, lactoferrin, myeloperoxidase, total salivary peroxidase, and thiocyanate) and immunologic (total immunoglobulin A, immunoglobulin G, and immunoglobulin M) mucous host defense factors were determined for paraffin-stimulated whole saliva before and 1, 3, 6, 12, 24, and 48 h after drug administration. Glycopyrrolate serum concentrations were determined before and 2, 4, 6, 10, 15, and 30 min and 1, 2, 3, 6, 12, and 24 h after IV drug injection. Salivary flow rates were decreased significantly for 12 h after glycopyrrolate injection, compared with saline injection. The concentrations of immunologic and nonimmunologic defense factors were increased in the glycopyrrolate group, and differences between the groups were found for all factors (P < 0.05–0.001) except lysozyme and total salivary peroxidase. In contrast, because of the reduced flow rate, the output of all defense factors into the saliva was decreased after glycopyrrolate injection, compared with saline injection. Glycopyrrolate thus decreases the output of salivary host defense factors into the oral cavity.

Implications: Glycopyrrolate induces long-lasting hyposalivation and decreases the secretion of salivary immunologic and nonimmunologic defense factors in healthy volunteers.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Glycopyrrolate is a quaternary anticholinergic drug that is widely used in anesthesiology as a premedication and for the reversal of neuromuscular block (1). The inhibition of salivary secretion by glycopyrrolate is remarkable and long-lasting (2). In our previous studies (3,4), we observed appreciable hyposalivation after anesthesia and surgery, in association with changes in oral host defense systems. In this study, we analyzed the effects of glycopyrrolate on the oral mucous host defense capacity in healthy subjects. Our hypothesis was that glycopyrrolate might impair oral mucous host defenses postoperatively.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Twelve healthy male volunteers (age 21–29 yr, weight 63–82 kg, height 171–191 cm) participated in the study. The protocol for the study was approved by the Joint Ethics Committee of Turku University and Turku University Central Hospital. The Finnish National Board of Health was notified before the beginning of the study, according to national regulations. Before the study, the health of the subjects was ascertained by using detailed medical histories and clinical examinations. Written, informed consent was obtained from each subject. All volunteers were students with good oral health. The oral mucosa was carefully inspected by the same doctor, and no ulcers or inflammation was found.

The study had a double-blinded, cross-over design, with balanced randomization. Single IV injections of saline (placebo) and glycopyrrolate (4 µg/kg), of equal volumes, were given to each subject in randomized order, at 3-wk intervals. A nurse who was not otherwise participating in the study was responsible for study drug preparation. Two veins of each subject were cannulated, i.e., a dorsal vein of the hand for drug administration and the contralateral antecubital vein for collection of blood samples. The volunteers spent 7 h in the clinical laboratory. After sampling, they were allowed to return home and were advised to avoid physical exercise. A standard lunch was served 4 h after drug administration. The use of any other drugs was prohibited for 2 days before and after the study day.

Paraffin-stimulated whole saliva (3 mL) was collected before the IV injection and 1, 3, 6, 12, 24, and 48 h thereafter. The collection was performed as described in our previous studies (3,4), by having the subjects chew a 1-g piece of paraffin wax for 3 min. During the collection, all saliva was drooled into a graduated cylinder once or twice each minute; the collected volume was measured with an accuracy of 0.1 mL. To avoid any loss of saliva through swallowing, the patients were given detailed instructions, the collection was performed in a relaxed position in a normal chair, and the collection was always supervised by the same investigator (MTL). The collection time was recorded, and the secretion rate was determined as milliliters per minute. The subjects were not allowed to eat, drink, or smoke for 1 h before collection. The samples were kept on ice and taken immediately for analysis. A 100-µL aliquot of uncentrifuged saliva was used for a lysozyme assay. The rest of each sample was centrifuged (10 min, 18,000g, 4°C) and stored frozen at -70°C until measurement of total protein, thiocyanate, lactoferrin, and total immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations, as well as amylase, myeloperoxidase, and total salivary peroxidase activities.

All samples were thawed and analyzed within 3 mo. Amylase activity was determined by using the Phadebas method (Pharmacia, Uppsala, Sweden). Total protein concentrations were determined by using a colorimetric method (5). Thiocyanate ions were quantified by using the ferric nitrate method described by Betts and Dainton (6). Lysozyme levels were estimated by using Micrococcus lysodeikticus diffusion plates (lysozyme kit; Kallestad Laboratories, Chaska, MN), with lyophilized human urine lysozyme as a reference. Lactoferrin levels were determined with a noncompetitive avidin-biotin enzyme immunoassay (7). Human colostral lactoferrin (Sigma Chemical Co., St. Louis, MO), further purified by affinity chromatography, was used as a standard. The absorbances in the lactoferrin assay were measured by using an automatic spectrophotometer (Titertek Multiscan®; Eflab Oy, Helsinki, Finland). Salivary peroxidase activity was assessed at 22°C, by measuring the oxidation rate of 5-thio-2-nitrobenzoic acid to (5-thio-2-nitrobenzoic acid)2 by hypothiocyanite ions generated during the oxidation of thiocyanate ions by salivary peroxidase (8), as described earlier (9). Specific myeloperoxidase activity in human saliva was measured by replacing thiocyanate with chloride in the assay mixture, because chloride is oxidized by myeloperoxidase but not by salivary peroxidase (10). Salivary IgA, IgG, and IgM concentrations were assayed by using a "trapping antibody"-type enzyme immunoassay (11). In this method, immobilized isotype-specific antihuman immunoglobulins trap sample antibodies, which are then detected by enzyme-conjugated antibodies. Rabbit heavy chain-specific anti-IgA, -IgG, and -IgM and the corresponding reagents conjugated with horseradish peroxidase were from Dako-Immunoglobulins (Copenhagen, Denmark). The immunoglobulin standards were purified from human serum (Behringwerke AG, Marburg, Germany). The substrate for the enzyme immunoassay, 1,2-phenylenediamine, was purchased from Sigma.

Blood samples for measurement of glycopyrrolate concentrations were collected before and 2, 4, 6, 10, 15, and 30 min and 1, 2, 3, 6, 12, 24, and 48 h after IV injection of the drug. All samples were centrifuged (4000g), and the serum was stored at -70°C. A modified radioreceptor assay, as described elsewhere (12), was used for the determination of plasma glycopyrrolate concentrations. The analysis, based on competition between glycopyrrolate and tritiated N-methylscopolamine, measured antimuscarinic activity in the plasma sample after glycopyrrolate administration. In brief, glycopyrrolate extracted from plasma with dicloethane was incubated with muscarinic receptors from a rat brain membrane preparation, with tritiated ligand in the assay buffer. The bound ligand was separated from the incubation mixture by harvesting and, after the addition of scintillation liquid, counted in a scintillation counter. The detection limit was 0.25 ng of glycopyrrolate in 1 mL of plasma. No glycopyrrolate was found in any of the baseline samples. The area under the concentration versus time curve (AUC) for glycopyrrolate was calculated to infinity by using the trapezoidal rule, and total plasma clearance was calculated by using the formula doseIV/AUC. The distribution volume was calculated as doseIV/AUC x terminal elimination rate. The pharmacokinetic calculations were performed using TopFit® software (version 2.0; G. Fischer, VCH Publisher, New York, NY).

Statistical analysis was performed by using overall analysis of variance (general linear model) for repeated measurements with two within factors (drug and time). Student’s paired t-test was used as the post hoc test and for comparison of values between the groups at each time point. The results are shown as mean ± SEM. P values of <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The stimulated salivary flow rate decreased from 1.7 ± 0.2 to 0.2 ± 0.04 mL/min (P < 0.001) and recovered to the baseline level by 24 h after the injection of glycopyrrolate, whereas no such event was observed after saline injection. The difference between flow rates was significant (P < 0.01) as long as 12 h after glycopyrrolate administration (Fig. 1). The concentration of total protein (Fig. 2) and the concentrations/activities of amylase, myeloperoxidase, thiocyanate, IgG, and IgA (Fig. 3) were higher for 12 h after the injection of glycopyrrolate, compared with saline (P < 0.05–0.001). Similar differences were noted for lactoferrin levels for 6 h (P < 0.05–0.01) and IgM levels for 48 h (P < 0.05–0.01), but no difference between the injections was observed for lysozyme or total peroxidase activities (Fig. 3).



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  		Figure 1. Glycopyrrolate concentrations in serum (S-glycopyrrolate) and salivary flow rates for the glycopyrrolate and placebo groups. **P < 0.01, ***P < 0.001 for the differences in salivary flow rates between the groups. Error bars were omitted for clarity.

 


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  		Figure 2. Concentrations of total salivary protein in the glycopyrrolate ({square}) and placebo ({blacksquare}) groups. *P < 0.05, ***P < 0.001 for the differences between the groups.

 


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  		Figure 3. Concentrations of salivary nonimmunologic and immunologic defense factors in the glycopyrrolate ({square}) and placebo ({blacksquare}) groups. *P < 0.05, **P < 0.01, ***P < 0.001 for the differences between the groups.

 
In contrast, the secretion rate of total protein and that of immunologic and nonimmunologic defense factors decreased for 1–12 h after glycopyrrolate injections, compared with saline injections (P < 0.05–0.001). Lactoferrin was an exception, with a statistically nonsignificant difference (Table 1). Compared with baseline values, the secretion rates of all host defense factors generally decreased after the injection of glycopyrrolate but increased after the injection of saline.


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  		Table 1. Secretion Rates of Salivary Total Protein and Selected Nonimmunologic and Immunologic Defence Factors in the Glycopyrrolate and Placebo Groups
 
The peak plasma glycopyrrolate concentrations (4.92–19.35 ng/mL) were reached 2 min after injection in 11 subjects. The peak concentration was observed after 4 min in one subject. The concentrations were less than the detection limit of 0.25 ng/mL in seven subjects after 2 h, and only two subjects had measurable drug concentrations 3 h after glycopyrrolate injections. The mean half-life of glycopyrrolate in the elimination phase was 34.9 ± 4.4 min. Its AUC was 151.6 ± 17.6 ng · min-1 · mL-1, the clearance was 1.8 ± 0.2 L · h-1 · kg-1, and the distribution volume was 1.4 ± 0.1 L/kg.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Hyposalivation is an important dysfunction of the salivary glands. Glycopyrrolate induces a decrease in salivary function through its parasympatholytic activity, blocking muscarinic receptors (13). Earlier studies found that the antisialologic effects of glycopyrrolate lasted for six hours after IM injection of 0.4 mg in adult volunteers (2) and up to eight hours after IM injection of 8 µg/kg in patients undergoing hysterectomies (14). However, no statistically significant antisialogogic effect of 8 µg/kg IM glycopyrrolate could be observed in elderly patients undergoing ocular surgery (15). In these earlier studies, the antisialogogic effects of glycopyrrolate were compared with baseline values, without control groups. Moreover, Ali-Melkkilä et al. (15) measured the antisialogogic effects using a visual analog scale (VAS) and only in small patient groups. In these earlier studies, glycopyrrolate was administered IM and the follow-up time did not exceed eight hours (2,14,16). The pharmacokinetics of glycopyrrolate in this study agree well with results reported for young parturients after IM glycopyrrolate (17), but somewhat lower distribution volumes and total clearances were observed for elderly patients scheduled for ocular surgery (15).

To our knowledge, the present study is the first to quantify accurately the antisialogogic effect of glycopyrrolate, by measuring salivary flow rates and by using a control group. The dosage used, 4 µg/kg IV glycopyrrolate, is representative of clinical dosages. In this study, the antisialogogic effect of glycopyrrolate was seen for 12 hours after IV drug injection, and normal salivation recovered in 24 hours. Earlier, we studied the effects of open-heart and major surgery on oral mucous host defense systems (3,4), and we considered glycopyrrolate a possible cause of the observed changes. On the basis of the present findings, a contribution of glycopyrrolate to the changes seen in host defense systems during the first postoperative days seems plausible.

Various methods have been used to study drug-induced decreases in salivary flow rates. The changes in salivation have been estimated subjectively by patients’ using a VAS. In this study, as in our previous studies (3,4), paraffin-stimulated whole saliva was collected. We also included a control group, which is obviously important when measuring the antisialogogic effect of a drug. There is a correlation between subjective assessments and stimulated whole-saliva flow rates. In cases of relatively mild symptoms of a dry mouth, the unstimulated whole-saliva flow rate is even more sensitive than the stimulated rate (18). However, the antisialogogic effect of glycopyrrolate is convincing, even 5 times more potent than that of atropine (2); therefore, the decrease in stimulated salivary flow rates is also reflected in high VAS scores.

After placebo treatment, the salivary flow rates increased slightly during the day. It is known from longitudinal studies that subjects tend to secrete more saliva as they become accustomed to the collection procedure (19). On the other hand, salivary flow rates vary at different times of the day. Circadian variation also occurs in the concentrations of salivary proteins (20), with the highest protein concentrations occurring late in the afternoon. This intraday variation was particularly remarkable in the concentrations of amylase, myeloperoxidase, and total salivary peroxidase in our study. The increased concentration of amylase in both of our groups may be attributable to direct stimulation of the parotid glands, because salivary amylase is almost exclusively derived from the parotid glands (21). The increase in total protein most likely reflects the chewing-stimulated output of salivary gland-synthesized proteins, of which amylase is the most abundant. However, total salivary peroxidase is the sum of leukocyte-derived myeloperoxidase and salivary peroxidase from the major salivary glands. Therefore, the increases in amylase and total salivary peroxidase activities are possibly attributable to stimulation by paraffin chewing. However, myeloperoxidase is not synthesized in the salivary glands, and increased myeloperoxidase activity in whole saliva thus indicates the influx of polymorphonuclear leukocytes or serum proteins into the saliva (22).

Although the salivary concentrations of both immunologic and nonimmunologic defense factors were increased after glycopyrrolate injections in our study, their secretion per time (output) decreased as a result of the strongly reduced salivary flow rate. Hyposalivation is known to diminish the output of antimicrobial factors (23). The present results paralleled the findings in our previous studies (3,4), except regarding the output of immunoglobulins. The secretion of IgG and IgM increased after open-heart surgery, but the secretion of IgG decreased, with no IgA or IgM alterations, after hysterectomies. The disparity in the results may be explained by the operations themselves and their effects on salivary flow rates or by individual variations in immunoglobulin secretion. In this study, the output of immunoglobulins corresponded to that of total protein. After the injection of glycopyrrolate, a difference in the durations of diminished immunoglobulin secretion was also seen. The decreased IgG secretion lasted for at least six hours, but there were no differences in the outputs of IgA and IgM three hours after injections. The different origins of salivary immunoglobulins may explain this. IgG is derived from serum, whereas most IgA and IgM antibodies are produced locally (24).

The hyposalivation induced by glycopyrrolate causes marked and prolonged impairment of the secretion of oral host defense factors. This can be harmful to patients who have a systemic disease or condition related to hyposalivation. Decreased salivation can also increase the occurrence of oral diseases and make patients susceptible to mucositis, especially candidiasis (25). This calls into question the value of the routine use of glycopyrrolate as anesthesia premedication and even for the reversal of neuromuscular block in these patients.


   Acknowledgments
 
This work was supported by grants from the Academy of Finland and Turku University Central Hospital.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Mirakhur RK, Dundee JW. Glycopyrrolate: pharmacology and clinical use. Anaesthesia 1983;38:1195–204.[ISI][Medline]
  2. Mirakhur RK, Dundee JW. Comparison of the effects of atropine and glycopyrrolate on various end-organs. J R Soc Med 1980;73:727–30.[ISI][Medline]
  3. Lähteenmäki M, Tenovuo J, Salo M, Perttilä J. Effects of open heart surgery on oral mucous host defence systems. Acta Anaesthesiol Scand 1997;41:837–42.[ISI][Medline]
  4. Lähteenmäki M, Salo M, Tenovuo J. Mucosal host defense response to hysterectomy assessed by saliva analyses: a comparison of propofol and isoflurane anaesthesia. Anaesthesia 1998;53:1067–73.[ISI][Medline]
  5. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.[Free Full Text]
  6. Betts RH, Dainton FS. Electron transfer and other processes involved in the spontaneous bleaching of acidified solutions of ferric thiocyanate. J Am Chem Soc 1953;75:5721–7.
  7. Vilja P, Krohn K, Tuohimaa P. A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin. J Immunol Methods 1985;76:73–83.[ISI][Medline]
  8. Wever R, Kast WM, Kasinoedin JH, Boelens R. The peroxidation of thiocyanate catalysed by myeloperoxidase and lactoperoxidase. Biochim Biophys Acta 1982;709:212–9.[Medline]
  9. Tenovuo J, Lehtonen OP, Aaltonen AS, et al. Antimicrobial factors in whole saliva in human infants. Infect Immun 1986;51:49–53.[Abstract/Free Full Text]
  10. Månsson-Rahemtulla B, Baldone DC, Pruitt KM, Rahemtulla F. Specific assays for peroxidases in human saliva. Arch Oral Biol 1986;31:661–8.[ISI][Medline]
  11. Lehtonen OP, Gråhn E, Ståhlberg T, Laitinen LA. Amount and avidity of salivary and serum antibodies against Streptococcus mutans in two groups of human subjects with different dental caries susceptibility. Infect Immun 1984;43:308–13.[Abstract/Free Full Text]
  12. Kaila T, Ali-Melkkilä T, Iisalo E, Kanto J. Radioreceptor assay for pharmacokinetic studies of glycopyrrolate. Pharmacol Toxicol 1990;67:313–6.[ISI][Medline]
  13. Schubert MM, Izutsu KT. Causes of salivary gland dysfunction. J Dent Res 1987;66:680–8.
  14. Ali-Melkkilä T, Kaila T, Kanto J, Iisalo E. Pharmacokinetics of i.m. glycopyrronium. Br J Anaesth 1990;64:667–9.[Abstract/Free Full Text]
  15. Ali-Melkkilä T, Kaila T, Kanto J. Glycopyrrolate: pharmacokinetics and some pharmacodynamic findings. Acta Anaesthesiol Scand 1989;33:513–7.[ISI][Medline]
  16. Wyant GM, Kao E. Glycopyrrolate methobromide. 1. Effect on salivary secretion. Can Anaesth Soc J 1974;21:230–41.[ISI][Medline]
  17. Ali-Melkkilä T, Kaila T, Kanto J, Iisalo E. Pharmacokinetics of glycopyrronium in parturients. Anaesthesia 1990;45:634–7.[ISI][Medline]
  18. Wang SI, Zhao ZT, Li J, et al. Investigation of the clinical value of total saliva flow rates. Arch Oral Biol 1998;43:39–43.[ISI][Medline]
  19. Kirstilä V, Tenovuo J, Ruuskanen O, et al. Longitudinal analysis of human salivary immunoglobulins, nonimmune antimicrobial agents and microflora after tonsillectomy. Clin Immunol Immunopathol 1996;80:110–5.[ISI][Medline]
  20. Dawes C, Chebib FS. The influence of previous stimulation and the day of the week on the concentrations of protein and the main electrolytes in human parotid saliva. Arch Oral Biol 1972;17:1289–301.[ISI][Medline]
  21. Paxton JW. Measurements of drugs in saliva: a review. Methods Find Exp Clin Pharmacol 1979;1:11–21.[Medline]
  22. Tenovuo J. Nonimmunoglobulin defense factors in human saliva. In: Tenovuo J, ed. Human saliva: clinical chemistry and microbiology. Vol II. Boca Raton, FL: CRC Press, 1989:55–92.
  23. Mandel ID. The functions of saliva. J Dent Res 1987;66:623–7.
  24. Brandzaeg P. Salivary immunoglobulins. In: Tenovuo J, ed. Human saliva: clinical chemistry and microbiology. Vol II. Boca Raton, FL: CRC Press, 1989:1–54.
  25. Närhi TO, Meurman JH, Ainamo A. Xerostomia and hyposalivation: causes, consequences and treatment in the elderly. Drugs Aging 1999;15:103–16.[ISI][Medline]
Accepted for publication April 21, 2000.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press