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Beta-glucuronidase studies in women
BETA-GLUCURONIDASE III.
Trichomonas
Vaginalis
STUDIES
Vaginitis
and Vaginal
IN WOMEN Fluid Enzyme Activity*
S. C. KASDON, M.D., J. MCGOWAN, F. HOMBUFWER, (From
M.D., W. H. FISHMAN, M.D., BOSTON, MASS.
Cancer Research and Cancer Control Unit Departments of Medicine and Biochemistry,
the
of
Tufts
PH.D.,
AND
the Department of Surgery, College Medical School)
and
the
T
HE study of beta-glucuronidase activity in vaginal fluid has been under way for some time11 2, 3 and an important aspect of this research program has been the clarification of the role this enzyme plays in the vaginal fluid. A discussion at length of the possible physiologic sources of the enzyme in vaginal fluid has been presented. This present report takes up in some detail the part played by Trichomonas infestation in the production of the beta-glucuronidase activity of vaginal fluid not associated with genital malignancy or pregnancy.4 The suggestion had been offered’ that Trichomonas vaginitis and pregnancy make up the principal sources of high vaginal fluid enzyme activity in patients free from genital malignancy. The occurrence of low beta-glucuronidase assays in the vaginal fluid of some patients with motile Trichomonas found in the hanging-drop preparation of the vaginal fluid casts doubt upon any direct relationship between the two. An earlier report1 demonstrated the lack of beta-glucuronidase activity in active pure cultures of Trichomonas organisms and the lysed organisms themselves. The detection ol! traces of beta-glucuronidase activity in various bacterial culture9 is dependent upon menthol-glucuronic acid in the medium., an unphysiologic compound. The beta-glucuronidase activity of pure cultures of Trichomonas in Kupferberg’s medium6 can be attributed to the enzyme present in the serum used in preparation of the culture medium. A systematic approach to the elucidation of the part played by Trichomonas infestation in the vaginal fluid is considered essential to the understanding of the source of beta-glucuronidase in this fluid. Other pdssible sources of the enzyme in the vaginal canal have been cnnsjdered in an earlier publication” : the frequent19 7 presence of high enzyme act,ivity in the blind vaginal pouch which remains following panhysterectomy in women helps to clarify its source. Certainly, the cervix and its secretion, the endomctrial cavity and its cellular fluid play no part in the elaboration of ~leta-gluclxronidase in vaginal fluid when the entire uterus has been eliminated. Since it has been suggested* that cultural methods for isolation of Trichomonas protozoa are at least as good and probably better than the hanging-drop *This study American Cancer Cancer Research,
was aided by Society, Inc., New York.
grants New
from York.
the and
647
United States Public the Damon Runyon
Health Memorial
Service fini
!@
Material
and Method
The vaginal fluids of 1% women were analyzed for: ( I 1 beta-~ln~n~orlidilst~ activity, (2) the presence of motilta Tricbomonas organisms on hanging-drop preparation, and (3) culture for Trichomonas in Kupferberg’s medium. The enzyme analysis was carried out as desrribecl earlier bj. Fishman and (‘o-workers. The hanging-drop preparation was ma(h) 1)~ inserting a sterile swab high into the vaginal vault without the use of Iul)Cat ion or antiseptics and immersing it direct,ly into sterile warm saline ill sla~~lar~l lest trtbes. The majority were checked for motile organisms wit hill Fori !--ti~c) minutch 01’ collection. I’lCN41 hanging-drop preparation was searched For not less than fire minutes before it was classed as negative for motile organisms. The r~nltnl~r medium and tech,Jackson, and Sprince.” Thr nique used were that, su ggcsted b.v linpferberg, medium was freshly prepared for t,hr vaginal fluids studied. The enltnres were oxamined for grou th of motile Trichomomls organisms at t.wcnty-four to seventytwo hours after inoculat.ion. An additional 41 women with Trichomonas vaginitis previously demonstrated by hanging drop or Pa.panicolaou sn1ea.1~WWP (evaluated for vaginal fluid betaglucuronidase activity. In both groups. specimens were obtained simultaneously for trichomonad population and enzyme itc?ivit>-.
Results and Discussion Two studies were c*ompleted: one on women known to have Trichornonas infestations and t,he ot,her on an unselectrtl group of WOI~WII. In the first, group, a total of forty-olte WUmeii wit.h l’richomonws vaginitis identified I)y hangin~drop preparation or Papanicolaoa smear were studied for beta-glucuronidase activit,v in tho vaginal fluid (Table I ) . Multiple specirnens were obtained in some of them and each assay was given equal value in analysis of the data. None of the womert were under treatment for Trichomonas infe&ion. The majority of t,hese 41 women had symptoms of vaginitis which The Fubjects were classified arbitrarily brought them into the present study. at. values above or below 300 units per gram of beta-glucuronidasr since this level has been suggested by other+ ” as critical in the interpretation of enzyme values for diagnostic purposes. Three patients (Table 11) with motile Trichomonas organisms in the vaginal fluid were found to hare multiple values both above and below 300 units per gram. One subjecat (No. 1) participating in :I cyclic vaginal fluid study had a total of fift,y-five separate determinat,ions with a range of values from 3 0 to 1,695 units per& pyarn. In t,his study, no attempt has been made to evaluate t,he factors oil ape and I lie menopause. In the second study, a group ol’ 96 wornu~~ untler GUT in one of three institutions” for miscellaneous diagnoses not connected with Triehomouas infestation Mass.,
*New and
England Center Hospital, Boston. Mass.. Holy Ghost Hospital, Cambridge, Mass.
Jewish
Memorial
Hospital,
Roxbury,
;$g”,
BETA-GLUCURONIDASE
STUDIES
IN
649
WOMEN
were investigated. Banging-drop preparations and cultures for the protozoa were carried out simultaneously with beta-glucuronidase activity assays (Table III). None of these patients were under treatment for Trichomonas vaginitis. It can be seen that previously unsuspected Trichomonas vaginitis was found in 7 per cent by hanging-drop preparation and in 9 per cent by the cultural method employed. In no case was a posit,ive hanging-drop preparation found without the organisms growing out in the culture medium. In two instances the culture was positive for Trichomonas organisms and careful study of hanging-drop preparations failed to reveal motile organisms. Jt was necessary to eliminate pregnant women and those with cancer of the cervix uteri in order to avoid confusion from them as sources of beta-glucuronidase in the vaginal fluid. I.
TABLE
TRICHOMONAS
VAGINITIS
AND BETA-GL~CITRONIDASE
ACTIVITY
IN VAGINAL
FLUID
-~ NUMBEROF
/3-GLUCURONIDASEACTIVITY (UNITS/GM.)
Under Over
TABLE
II.
300 300 Total VAGINITIS
With
Multiple
AND
1
2 3
NO. DETERMINATIONS
55 5 2
BELOW NUMBER
38 1 1
TRICHOMONAS VAGINITIS
SPECIMENS
17 40 57
BETA-GLUCURONIDASE
Specimens
-___ SUBJECT
/
14 27 41
TRICHOMONAS
Subjects
SUBJECTS
Demonstrating 300
UNITS/GM. 1
VALUES
20-300 51 138
+ +
ACTIVITY
High
and
IN
Low
ABOVE NUMBER
17 4 1
VAGINAL
FLUID
Values 300
UNITS/GM. /
VALUES
301-1695 301-2020 376
It is clear from Table III that values below 300 units of activity are essentially the same for women with hanging-drop (65 per cent) or cultural evidence (63 per cent) of Trichomonas protozoa in the vaginal fluid and for those (59 per cent) without evidence of Trichomonas infestation. The distribution of values above 300 units per gram of beta-glucuronidase activity shows no significant variation attributable to the presence or absence of Trichomonas organisms (Fig. 1). Women showing vaginal fluid beta-glucuronidase activities below 300 units per gram constituted 77 per cent of the total in the age group 20-40, 59 per cent in the group 41 to 60, and 43.5 per cent in the group 61 to 90 years of age.l In the present study, our figures of 64 and 59 per cent are in agreement with the previous data since 33 out of the 96 women mere postmenopausal and very few of the subjects were younger than 40 years old. The view has been offered by Ode11 and othcrsS that substances in vaginal fluid promote the elaboration of beta-glucnronidase enzyme from. vaginal microorganisms. This statement was based on the demonstration by Buehler and associates,10 that menthol glucuronide in the medium of growing Escherichia coli stimulated glueuronidase production. In support of this, Ode11 presented data showing that certain bacteria populating the vaginal tract were capable of producing beta-glucuronidase in the presence of menthol glucuronide. To test this hypothesis, a fresh, sterile sample of vaginal fluid was incubated in the presence of a multiplying strain of h’. coli for two weeks. In a companion culture, the same organisms were grown in culture medium containing menthol glucuronide. Whereas in this la.tter culture beta-glucuronidase production was
Hanging-drop posltlve Culture positive (:ult,nr6 and hanging-drop
P-GLUCI:BONIUASEACTIVIT~ UNITS/GM.
ii 58
II 11 95
i0 LO 45
201. 300
101. 200
oIt)11
301. 400 6 7 33 2 29
401500
Sol- 601. 6011 700 1 2 2 2 17 12
701. 800 1 1 9 3" 11
801. 900
YOlli 7
1000
1001-
2 2 15
1500
3 3 1
2000
1x1-
(i
ii
3
CJ
+
II 1
“501
“001-
“5Oll
6" 64 336
-si’G\rm
s
7 9 87
i;, li., i‘,“[‘$.
BETA-GI,UCURONIDASE
Volume 61
STUDIES
IN
651
WOMEN
stimulated, the culture containing vaginal fluid showed no increase in enzyme content. If one may draw any conclusion from this one experiment, there is little likelihood that substrates similar in their function to menthol glucuronide exist in sterile vaginal fluid. The evidence from our previous, present, and unpublished studies points to a relationship of beta-glucuronidase in the vaginal fhoid and factors of internal secretory origin. The mere presence of Trichomonas organisms in the vaginal fluid in women without complaint referable to vaginitis is of no significance in explaining high vaginal fluid beta-glucuronidase values. However, as we have seen in Table I, the clinical state of Trichomonas vaginitis is not infrequently Here, of course, it is difficult to associated with elevated beta-glucuronidase. evaluate the relative importance of the leucocyte population and tissue response to Trichomonas organisms. The part played by the leucocytes in the vaginal fluid enzyme activity is described in a separate study.‘l 40 -i
TRICHOMONAS SUBJECTS
t x-
-x
CONTROL SUBJECTS
I
PERCENTAGE OF SPECIMENS STUDIED
1
: :F 200 400 600 800 1,000 1,500 2,000 , -IVAGINAL FLUID P-GLUCURONIOASE
Fig.
l.-Relationship
between
the
presence activity
of Trichomonas of vaginal
UNITS PER GRAM
organisms fluid.
and
the
beta-glucuronidase
Summary A total of 96 nonpregnant, noncancerous healthy women over 40 years of age were studied for vaginal fluid beta-glucuronidase activity in its relation to Trichomonas infestation. Nine of the subjects had previously unsuspected Trichomonas o-rganisms identified by culture and seven of these were positive by examination of the hanging-drop preparation. Sixty-three per cent of 64 specimens in the Trichomonas group exhibited values for beta-glucuronidase activity below 300 units per gram. This compared closely with 59 per cent obtained in the women showing no Trichomonas organisms. In individual subjects with Trichomonas who were studied at intervals over a period of months values were
7. s. 9. IO. I 1.
,J. A. M. A. 143: 350, 1950. Fishman, W. H., Kasdon, S. C., and Homburgrr, F.: Kasdon, S. C., Fishman, W. II., and IIomburgc~, k’.: .J. A. M. h. 144: 892, 1950. Research 10: 016, 1950. Fishman, W. H., Kasdon, S. C., and Hamburger, E’.: (!ancer Odell, L. D., and Burt, J. C.: Science 109: 564, 1949. Odell, L. D., Priddle, H. D., and Burt, .J. (1.: &II. -1. Clin. Path. 20: 133, 1950. Proc. Sot>. Exper. HilrI. & Med. 67: Kupferberg, 8. B., .Johnsou, 8.. and Rprirrw. H.: 304, 1948. Fishman, W. H., Kasdon, H. G., Konner. (‘., k’inhmau. I,, \I’., and Homl~urger, F.: In preparation. Hoffman, M. H.: AM. .J. OBST. & UY~.EC. 60: 224, 1950. Cancer Research 9: 362, 1949. Odell, 1,. D., and Burt, .J. C.: l
ISAY
STATE
Koau
Trichomonas
Vaginalis
STUDIES
Vaginitis
and Vaginal
IN WOMEN Fluid Enzyme Activity*
S. C. KASDON, M.D., J. MCGOWAN, F. HOMBUFWER, (From
M.D., W. H. FISHMAN, M.D., BOSTON, MASS.
Cancer Research and Cancer Control Unit Departments of Medicine and Biochemistry,
the
of
Tufts
PH.D.,
AND
the Department of Surgery, College Medical School)
and
the
T
HE study of beta-glucuronidase activity in vaginal fluid has been under way for some time11 2, 3 and an important aspect of this research program has been the clarification of the role this enzyme plays in the vaginal fluid. A discussion at length of the possible physiologic sources of the enzyme in vaginal fluid has been presented. This present report takes up in some detail the part played by Trichomonas infestation in the production of the beta-glucuronidase activity of vaginal fluid not associated with genital malignancy or pregnancy.4 The suggestion had been offered’ that Trichomonas vaginitis and pregnancy make up the principal sources of high vaginal fluid enzyme activity in patients free from genital malignancy. The occurrence of low beta-glucuronidase assays in the vaginal fluid of some patients with motile Trichomonas found in the hanging-drop preparation of the vaginal fluid casts doubt upon any direct relationship between the two. An earlier report1 demonstrated the lack of beta-glucuronidase activity in active pure cultures of Trichomonas organisms and the lysed organisms themselves. The detection ol! traces of beta-glucuronidase activity in various bacterial culture9 is dependent upon menthol-glucuronic acid in the medium., an unphysiologic compound. The beta-glucuronidase activity of pure cultures of Trichomonas in Kupferberg’s medium6 can be attributed to the enzyme present in the serum used in preparation of the culture medium. A systematic approach to the elucidation of the part played by Trichomonas infestation in the vaginal fluid is considered essential to the understanding of the source of beta-glucuronidase in this fluid. Other pdssible sources of the enzyme in the vaginal canal have been cnnsjdered in an earlier publication” : the frequent19 7 presence of high enzyme act,ivity in the blind vaginal pouch which remains following panhysterectomy in women helps to clarify its source. Certainly, the cervix and its secretion, the endomctrial cavity and its cellular fluid play no part in the elaboration of ~leta-gluclxronidase in vaginal fluid when the entire uterus has been eliminated. Since it has been suggested* that cultural methods for isolation of Trichomonas protozoa are at least as good and probably better than the hanging-drop *This study American Cancer Cancer Research,
was aided by Society, Inc., New York.
grants New
from York.
the and
647
United States Public the Damon Runyon
Health Memorial
Service fini
!@
Material
and Method
The vaginal fluids of 1% women were analyzed for: ( I 1 beta-~ln~n~orlidilst~ activity, (2) the presence of motilta Tricbomonas organisms on hanging-drop preparation, and (3) culture for Trichomonas in Kupferberg’s medium. The enzyme analysis was carried out as desrribecl earlier bj. Fishman and (‘o-workers. The hanging-drop preparation was ma(h) 1)~ inserting a sterile swab high into the vaginal vault without the use of Iul)Cat ion or antiseptics and immersing it direct,ly into sterile warm saline ill sla~~lar~l lest trtbes. The majority were checked for motile organisms wit hill Fori !--ti~c) minutch 01’ collection. I’lCN41 hanging-drop preparation was searched For not less than fire minutes before it was classed as negative for motile organisms. The r~nltnl~r medium and tech,Jackson, and Sprince.” Thr nique used were that, su ggcsted b.v linpferberg, medium was freshly prepared for t,hr vaginal fluids studied. The enltnres were oxamined for grou th of motile Trichomomls organisms at t.wcnty-four to seventytwo hours after inoculat.ion. An additional 41 women with Trichomonas vaginitis previously demonstrated by hanging drop or Pa.panicolaou sn1ea.1~WWP (evaluated for vaginal fluid betaglucuronidase activity. In both groups. specimens were obtained simultaneously for trichomonad population and enzyme itc?ivit>-.
Results and Discussion Two studies were c*ompleted: one on women known to have Trichornonas infestations and t,he ot,her on an unselectrtl group of WOI~WII. In the first, group, a total of forty-olte WUmeii wit.h l’richomonws vaginitis identified I)y hangin~drop preparation or Papanicolaoa smear were studied for beta-glucuronidase activit,v in tho vaginal fluid (Table I ) . Multiple specirnens were obtained in some of them and each assay was given equal value in analysis of the data. None of the womert were under treatment for Trichomonas infe&ion. The majority of t,hese 41 women had symptoms of vaginitis which The Fubjects were classified arbitrarily brought them into the present study. at. values above or below 300 units per gram of beta-glucuronidasr since this level has been suggested by other+ ” as critical in the interpretation of enzyme values for diagnostic purposes. Three patients (Table 11) with motile Trichomonas organisms in the vaginal fluid were found to hare multiple values both above and below 300 units per gram. One subjecat (No. 1) participating in :I cyclic vaginal fluid study had a total of fift,y-five separate determinat,ions with a range of values from 3 0 to 1,695 units per& pyarn. In t,his study, no attempt has been made to evaluate t,he factors oil ape and I lie menopause. In the second study, a group ol’ 96 wornu~~ untler GUT in one of three institutions” for miscellaneous diagnoses not connected with Triehomouas infestation Mass.,
*New and
England Center Hospital, Boston. Mass.. Holy Ghost Hospital, Cambridge, Mass.
Jewish
Memorial
Hospital,
Roxbury,
;$g”,
BETA-GLUCURONIDASE
STUDIES
IN
649
WOMEN
were investigated. Banging-drop preparations and cultures for the protozoa were carried out simultaneously with beta-glucuronidase activity assays (Table III). None of these patients were under treatment for Trichomonas vaginitis. It can be seen that previously unsuspected Trichomonas vaginitis was found in 7 per cent by hanging-drop preparation and in 9 per cent by the cultural method employed. In no case was a posit,ive hanging-drop preparation found without the organisms growing out in the culture medium. In two instances the culture was positive for Trichomonas organisms and careful study of hanging-drop preparations failed to reveal motile organisms. Jt was necessary to eliminate pregnant women and those with cancer of the cervix uteri in order to avoid confusion from them as sources of beta-glucuronidase in the vaginal fluid. I.
TABLE
TRICHOMONAS
VAGINITIS
AND BETA-GL~CITRONIDASE
ACTIVITY
IN VAGINAL
FLUID
-~ NUMBEROF
/3-GLUCURONIDASEACTIVITY (UNITS/GM.)
Under Over
TABLE
II.
300 300 Total VAGINITIS
With
Multiple
AND
1
2 3
NO. DETERMINATIONS
55 5 2
BELOW NUMBER
38 1 1
TRICHOMONAS VAGINITIS
SPECIMENS
17 40 57
BETA-GLUCURONIDASE
Specimens
-___ SUBJECT
/
14 27 41
TRICHOMONAS
Subjects
SUBJECTS
Demonstrating 300
UNITS/GM. 1
VALUES
20-300 51 138
+ +
ACTIVITY
High
and
IN
Low
ABOVE NUMBER
17 4 1
VAGINAL
FLUID
Values 300
UNITS/GM. /
VALUES
301-1695 301-2020 376
It is clear from Table III that values below 300 units of activity are essentially the same for women with hanging-drop (65 per cent) or cultural evidence (63 per cent) of Trichomonas protozoa in the vaginal fluid and for those (59 per cent) without evidence of Trichomonas infestation. The distribution of values above 300 units per gram of beta-glucuronidase activity shows no significant variation attributable to the presence or absence of Trichomonas organisms (Fig. 1). Women showing vaginal fluid beta-glucuronidase activities below 300 units per gram constituted 77 per cent of the total in the age group 20-40, 59 per cent in the group 41 to 60, and 43.5 per cent in the group 61 to 90 years of age.l In the present study, our figures of 64 and 59 per cent are in agreement with the previous data since 33 out of the 96 women mere postmenopausal and very few of the subjects were younger than 40 years old. The view has been offered by Ode11 and othcrsS that substances in vaginal fluid promote the elaboration of beta-glucnronidase enzyme from. vaginal microorganisms. This statement was based on the demonstration by Buehler and associates,10 that menthol glucuronide in the medium of growing Escherichia coli stimulated glueuronidase production. In support of this, Ode11 presented data showing that certain bacteria populating the vaginal tract were capable of producing beta-glucuronidase in the presence of menthol glucuronide. To test this hypothesis, a fresh, sterile sample of vaginal fluid was incubated in the presence of a multiplying strain of h’. coli for two weeks. In a companion culture, the same organisms were grown in culture medium containing menthol glucuronide. Whereas in this la.tter culture beta-glucuronidase production was
Hanging-drop posltlve Culture positive (:ult,nr6 and hanging-drop
P-GLUCI:BONIUASEACTIVIT~ UNITS/GM.
ii 58
II 11 95
i0 LO 45
201. 300
101. 200
oIt)11
301. 400 6 7 33 2 29
401500
Sol- 601. 6011 700 1 2 2 2 17 12
701. 800 1 1 9 3" 11
801. 900
YOlli 7
1000
1001-
2 2 15
1500
3 3 1
2000
1x1-
(i
ii
3
CJ
+
II 1
“501
“001-
“5Oll
6" 64 336
-si’G\rm
s
7 9 87
i;, li., i‘,“[‘$.
BETA-GI,UCURONIDASE
Volume 61
STUDIES
IN
651
WOMEN
stimulated, the culture containing vaginal fluid showed no increase in enzyme content. If one may draw any conclusion from this one experiment, there is little likelihood that substrates similar in their function to menthol glucuronide exist in sterile vaginal fluid. The evidence from our previous, present, and unpublished studies points to a relationship of beta-glucuronidase in the vaginal fhoid and factors of internal secretory origin. The mere presence of Trichomonas organisms in the vaginal fluid in women without complaint referable to vaginitis is of no significance in explaining high vaginal fluid beta-glucuronidase values. However, as we have seen in Table I, the clinical state of Trichomonas vaginitis is not infrequently Here, of course, it is difficult to associated with elevated beta-glucuronidase. evaluate the relative importance of the leucocyte population and tissue response to Trichomonas organisms. The part played by the leucocytes in the vaginal fluid enzyme activity is described in a separate study.‘l 40 -i
TRICHOMONAS SUBJECTS
t x-
-x
CONTROL SUBJECTS
I
PERCENTAGE OF SPECIMENS STUDIED
1
: :F 200 400 600 800 1,000 1,500 2,000 , -IVAGINAL FLUID P-GLUCURONIOASE
Fig.
l.-Relationship
between
the
presence activity
of Trichomonas of vaginal
UNITS PER GRAM
organisms fluid.
and
the
beta-glucuronidase
Summary A total of 96 nonpregnant, noncancerous healthy women over 40 years of age were studied for vaginal fluid beta-glucuronidase activity in its relation to Trichomonas infestation. Nine of the subjects had previously unsuspected Trichomonas o-rganisms identified by culture and seven of these were positive by examination of the hanging-drop preparation. Sixty-three per cent of 64 specimens in the Trichomonas group exhibited values for beta-glucuronidase activity below 300 units per gram. This compared closely with 59 per cent obtained in the women showing no Trichomonas organisms. In individual subjects with Trichomonas who were studied at intervals over a period of months values were
7. s. 9. IO. I 1.
,J. A. M. A. 143: 350, 1950. Fishman, W. H., Kasdon, S. C., and Homburgrr, F.: Kasdon, S. C., Fishman, W. II., and IIomburgc~, k’.: .J. A. M. h. 144: 892, 1950. Research 10: 016, 1950. Fishman, W. H., Kasdon, S. C., and Hamburger, E’.: (!ancer Odell, L. D., and Burt, J. C.: Science 109: 564, 1949. Odell, L. D., Priddle, H. D., and Burt, .J. (1.: &II. -1. Clin. Path. 20: 133, 1950. Proc. Sot>. Exper. HilrI. & Med. 67: Kupferberg, 8. B., .Johnsou, 8.. and Rprirrw. H.: 304, 1948. Fishman, W. H., Kasdon, H. G., Konner. (‘., k’inhmau. I,, \I’., and Homl~urger, F.: In preparation. Hoffman, M. H.: AM. .J. OBST. & UY~.EC. 60: 224, 1950. Cancer Research 9: 362, 1949. Odell, 1,. D., and Burt, .J. C.: l
ISAY
STATE
Koau