British Journal of Obstetrics and Gynaecology October 1978. Vol85. pp 77G782
CHANGES IN SALIVARY PEROXIDASE AND POLYMORPHONUCLEAR NEUTROPHIL LEUCOCYTE ENZYME ACTIVITIES DURING THE MENSTRUAL CYCLE BY
S. M. COCKLE AND
R. A. HARKNESS Division of Perinatal Medicine, Clinical Research Centre Watford Road, Harrow HA1 3UJ, UK
Summary An elevation in salivary peroxidase activity has been found about the time of ovulation in 14 menstrual cycles in a total of six women. This peak coincided with the ovulatory luteinizing hormone (LH) and oestrogen peak in the four cycles in which endocrine studies were performed. Rises in some polymorphonuclear neutrophil leucocyte enzymes were also seen around ovulation. The possible use of changes in salivary peroxidase as a method for detection of ovulation is discussed.
in the human endometrium (Lucas et al, 1964) and peroxidase in bovine milk (Kern et al, 1963) show elevated activities about the time of ovulation. A fall in human milk peroxidase activity has been seen after delivery (Gothefors and Marklund, 1975) which may follow closely the rapid oestrogen withdrawal over the same period. Finally, physiological doses of oestrogen cause stimulation of uterine peroxidase activity in rats (Lyttle and Jellinck, 1972). We decided to investigate this effect of oestrogen using lactoperoxidase in human saliva as a model system, and report here the activities of peroxidase in salivary supernatant during the menstrual cycle in women. A second peroxidase, namely myeloperoxidase, was also studied about the time of ovulation, and changes compared with those found in salivary peroxidase. General enzymic effects were followed by studying other neutrophil enzymes.
PEROXIDASES are widespread and exist in a variety of molecular forms (Paul, 1963). They provide one of the pathways inside the cell for the breakdown of hydrogen peroxide, a reactive product of aerobic respiration (Fridovich, 1976), during the oxidation of a diverse range of substrates (Maehly and Chance, 1954). The distribution of peroxidases throughout the body fluids and tissues is highly variable (Lyttle and De Sombre, 1977), suggesting other physiological functions. For example, myeloperoxidase, in the polymorphonuclear neutrophil leucocyte (neutrophil) and lactoperoxidase found in saliva, milk and tears have bactericidal properties (Klebanoff, 1968; Hogg and Jago, 1970). Peroxidases may, therefore, play an important role in defence mechanisms against bacterial invasion. Investigations have indicated that oestrogens effect peroxidase production. Uterine peroxidase 776
SALIVARY PEROXIDASE DURING CYCLE
METHODS Clinical methods Volunteers (seven women and four men) collected 1 ml of saliva, before breakfast, in a universal container. The secretion of saliva and its composition vary considerably in a response to a variety of physiological circumstances, and because of this, the importance of collecting the samples a t the same time of day, before eating, and from the same part of the mouth was stressed. Samples, stored at 4 "C for up to five days, were centrifuged at 1500 g for 20 minutes and the supernatant retained for peroxidase and protein determinations. Salivary peroxidase activity is expressed relative to protein in preference to volume, as the water content of saliva varies considerably. Salivary peroxidase is stable for at least one week at 4 "C. A 20 ml sample of heparinized venous blood was collected daily from women at about the time of ovulation for neutrophil studies and estimation of plasma hormone levels. Approval for blood samples was obtained from the Brent and Harrow Health Authority Ethical Committee. Biochemical methods All materials were of analytical quality or of the highest purity available. 2,T-Azino di (3-ethyl benzothiazoline-6-sulphonicacid) ABTS, was obtained from Boehringer (Mannheim, Germany), and 6 per cent Dextran of molecular weight 110 000 in 0.9 per cent saline (Dextraven) from Fisons Ltd (Loughborough, Leicestershire, England). All other substrates, enzymes and reagents were obtained from Sigma Chemical Co (London, England). Peroxidase assay The assay was performed, using the chromogen ABTS, as described by Shindler et a1 (1976) with the modifications that the reaction was carried out at 25 "C in a final volume of 3 ml. The assay medium contained 1 mM ABTS and 0.1 mM hydrogen peroxide in 0.1 M sodium acetate buffer pH 4.4. The absorbence change was followed at 412 nm in a cuvette of 1 cm path length using a Unicam SP 1800 Spectrophotometer. The oxidation of one pmol of ABTS under the conditions of the assay
777
leads to a change in absorbence of 32.4. Units for all enzyme activities in this report are expressed as pmol of substrate utilized/ minute per g of protein. Total proteins were measured by the method of Miller (1959) using bovine serum albumin as the standard protein. Preparation of blood samples from women at about the time of ovulation A 20 ml sample of heparinized venous blood, collected daily around the time of ovulation, was centrifuged at 2000 g for five minutes. The blood cells were resuspended in isotonic saline and the neutrophils separated by a modification of the method of Lehrer (1971). Six per cent dextran in 0 - 9 per cent saline was added in the ratio three volumes dextran to ten volumes red cell suspension, and the erythrocytes allowed to sediment for 100 minutes at Ig. The neutrophil-rich supernatant was centrifuged at 2000 g for five minutes and the pellet retained. The cells were treated with 0.87 per cent (w/v) NH4CI to cause the lysis of contaminating erythrocytes, and then washed repeatedly in isotonic glucose until free from haemoglobin and red cell ghosts. The cells were resuspended in 1 ml of isotonic glucose, lyzed by freezing and thawing three times, and finally assayed for Pperoxidase, alkaline phosphatase, glucuronidase, acid phosphatase and total protein. Enzyme assays in neutrophils and saliva Alkaline phosphatase was assayed by the method of Hausamen et a1 (1967); acid phosphatase by the method of Mitchell et a1 (1970); and P-glucuronidase by the method of Fishman et a1 (1967). Arylsulphatase was assayed after removal of inhibitory phosphate ions from the saliva, according to the method of Boyer and France (1976). N-acetyl-P-D-glucosaminidase in saliva was assayed using a method based on those described by Borooah et al (1961) and Woollen et a1 (1961). The assay medium, of final volume 1 ml, contained 3 - 6 mM p-nitropheny1-Nacetyl-8-D-glucosaminide in 0.05 M citrate buffer pH 4.5, and an aliquot of saliva to
778
COCKLE AND HARKNESS
initiate the reaction. After incubation for 30 minutes at 37 “C, the reaction was stopped by addition of 2 ml of 0 . 2 M borate buffer (pH 9.8) and the absorbance at 405 nm was read against a suitable blank.
z.-
-
400 ;cul]
-
.-k
100
E E,
x
3
5
7
9
11 13 15 17 19 21 23 25 1
1 3
5
7
9
11 13 15 17 19 21 23 1 Day of cycle
1
0
RESULTS Salivary peroxidase activities Serial peroxidase activities in the saliva of seven women (ages 18 to 45) were studied throughout 15 menstrual cycles. All showed mid-cycle peaks in activity, with the exception of one from whom samples were not available over the ovulatory period. Ninety-nine per cent confidence limits for peroxidase activity were calculated for all cycles. Eight consecutive activities, during either the early follicular or late luteal phases, were used to calculate the mean and its confidence limits. With the exception of one woman, B, the mid-cycle peaks of peroxidase activity lay outside these confidence limits. Peroxidase activities throughout five complete menstrual cycles are shown in Figures 1,2 and 3. The magnitude of the mid-cycle peak is variable, showing about a two-fold rise in three of these cycles (subject A) and about a three-fold rise in subjects C and D. The peak is considerably smaller in subject B (Fig. 6 ) . Two consecutive cycles from one woman, A, show repeatable patterns of peroxidase activity throughout
7s”^.ryl;,
300
ze
200
I?
100
-
FIG.2 Salivary peroxidase activities throughout two consecutive menstrual cycles in one woman, A. The solid bars represent menstruation.
1 4 5 0 1 Subject C
E
50
3
5
7
9 11 13 15 17 19 21 23 2 Day of cycle
4
FIG.3 Salivary peroxidase activities during one cycle in one woman, A. Both early morning (0730) and late evening (2300) saliva samples were studied. Total oestrogen excretion per 24 hours is shown over the ovulatory period. The solid bars represent menstruation.
;350
.-
3
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
15 17 19 21 23 25 27 29 31 33 35 37 39 41 431 3 500
Subject
D
f bysler) immun.
._
2
P
200
100 1
3
5
7
9 11 13 15 17 19 21 23 25 2 4 Day of cycle
FIG.1 Salivary peroxidase activities during one menstrual cycle in two women, C and D. The solid bars represent menstruation.
the cycle, as well as clear peaks at mid-cycle. The results in Figure 3, also from subject A, show that serial activities at 0730 and 2300 hours both have a clear increase at mid-cycle but all activities are about two to three times higher in the evening. The results from 14 cycles are superimposed as a scatter diagram (Fig. 4). The cycle in which data were not available over the ovulatory period was omitted. The time-scale refers to the number of days before menstruation
SALIVARY PEROXIDASE DURING CYCLE
779
- -
u
Cold
Subject H
600
n
1
26 24 22 20 18 16 14 12 10 8 6 4 Time in days before last menstruation
2
FIG.4 Accumulated peroxidase activities from 14 menstrual cycles. Peroxidase activity is expressed as a multiple of a mean value which is calculated from eight consecutive values from either the early follicular or late luteal phases of the cycle.
because the luteal phase tends to be more constant than the follicular phase of the menstrual cycle. Most points lie within a broad band of activity but over a mid-cycle period (days 11 to 19) eight points lie clear of this band with activities two to three times the mean. Serial peroxidase activities in the saliva of four men were studied throughout one month (Fig. 5). In contrast to the results for women, there is no common pattern. Peroxidase activities, in subjects E and F, vary only slightly throughout the month while G and H show marked elevations on certain days. After primary immunization, against tetanus toxoid and poliovirus of subject C (Fig. 1) and of a second woman B (not illustrated), there was a distinct elevation of salivary peroxidase activity over five days, followed by a rapid fall to previous levels. Subject D who only received a 'booster' dose of tetanus toxoid and poliovirus did not show this elevation. In the man, G, an allergic reaction to animals coincided with an elevation of salivary peroxidase activity, and an upper respiratory tract infection, in H, also appeared to elevate the activity.
3
5
7
9
11 13 I5 17 19 21 23 25 27 Day of month
FIG.5 Salivary peroxidase activities throughout one month in four men.
Relation to ovulation Endocrine data have confirmed that the mid-cycle peak of peroxidase coincides with elevations of plasma oestradiol, determined by radioimmunoassay, in three cycles (Figs. 6 , 7 and 8) and of total urinary oestrogens, determined fluorimetrically, in one cycle (Fig. 3). The plasma luteinizing hormone (LH) peak, determined by radioimmunoassay, occurs within one day of the peroxidase peak. Elevations in urinary pregnanediol, determined by gas liquid chromatography, after the peroxidase peak, confirmed the presence of a functioning corpus luteum in subjects A and B. Polymorphonuclear neutrophil Ieucocyte enzyme activities A selection of neutrophil enzyme activities were studied over the ovulatory period in three cycles (Figs. 6 , 7 and 8). In general, the patterns of enzyme activities in neutrophils are less clear than those for saliva as it was not practicable to obtain data throughout the whole month. However, elevations in neutrophil peroxidase, alkaline phosphatase and P-glucuronidase activities do appear to occur concurrently with rises in plasma oestradiol-17P in two women (Figs. 6 , 7 and 8). DISCUSSION A mid-cycle peak in salivary peroxidase which coincides with the ovulatory oestrogen
780
COCKLE AND HARKNESS I
2000
1 Cycle 3
I
I
Plasma Oestradiol
500
- 17 p
-
500
15w
' '"1
I
Leuc. Peroxidase
+ y c .
Alk. Phos.
:::1 0.9
0
- 3 - 2 - 1 0
1
2
3
4
Days before a n d after LH peak
FIG.6 Salivary peroxidase and neutrophil peroxidase and alkaline phosphatase activities around the time of ovulation in subject B (cycle 3). Enzyme activities and plasma oestradiol-l7,G are plotted relative to the LH peak.
Plasma Oeslradiol-176
Saliva Peroxidase
j
'"1 +
-Glucuronidase
'.j & 0
2w 1
Leuc. Alk. Phos
0
-5 -4 - 3 -2 -1 0 1 2 3 4 Days before and after LH peak
FIG.7 Salivary peroxidase and neutrophil peroxidase, ,G-glucuronidase and alkaline phosphatase activities around the time of ovulation in subject B (cycle 4). Enzyme activities and plasma oestradiol-179 are plotted relative to the LH peak.
w - G l u c u r o n i d a s e
,,
-id
Phosphatase
0 -3 - 2 -1 0 1 2 3 Days before and after LH peak
FIG.8 Salivary peroxidase and neutrophil peroxidase, alkaline phosphatase, p-glucuronidase and acid phosphatase activities around the time of ovulation in subject A (cycle 3). Enzyme activities and plasma oestradiol-l7,G are plotted relative to the LH peak.
peak has been found in all women from whom samples were available over the ovulatory period. The effects were repeatable in consecutive cycles in the same woman, and the peaks were of comparable magnitude, although their size varied markedly in different women. There was a marked contrast between the individual plots, and the scatter plot representation (Fig. 4). This was because of large variation between women. A peak shown clearly by an individual may be no higher than the generality of non-peak values shown by other women. Also, in the scatter plot a large variation in time of ovulation led to poor peak definition. Lactoperoxidase is possibly responsible for the change in salivary peroxidase activity seen at ovulation. It is the main peroxidase present in saliva, and is actively secreted from the salivary glands (Thomson and Morell, 1967). Myeloperoxidase, from neutrophils which migrate through the gingival crevice (Attstrom and Egelberg, 1970) and bacterial peroxidase are also present in saliva. Centrifugation of the saliva largely removed these cellular contaminants, although low concentrations of
SALIVARY PEROXIDASE DURING CYCLE
both peroxidases may be present in the supernatant due to lysis of the cells (Makinen and Tenovuo, 1976). The mid-cycle elevations in neutrophil enzyme activities, demonstrated here, are more difficult to interpret than those for salivary peroxidase. All activities are expressed per mg of total protein, and hence, are independent of the total number of cells in our preparation. It has been demonstrated that circulating neutrophil numbers increase around the time of ovulation in women (Bain and England, 1975), so that the elevations of neutrophil enzyme activities could be reflecting a proportional increase in neutrophil numbers within our white cell preparation. This cannot be the case as the preparation of cells used consisted of about 95 per cent neutrophils. Peaks in neutrophil alkaline phosphatase activities were found in this study, which are consistent with previous evidence for oestrogenic control of neutrophil alkaline phosphatase. For example, its activity is raised two to threefold during pregnancy and follows changes in oestrogen production (Polishuk et al, 1970). In addition, women aged between 19 and 48 have significantly higher neutrophil alkaline phosphatase activities than men, and the activities fall after the menopause (Rosner and Lee, 1965). The mid-cycle peak in salivary peroxidase activity could perhaps be used to detect ovulation. Saliva is a convenient and abundant source of peroxidase, and the assay is quick and sensitive (Table I). Previous workers have found peaks of N-acetyl-p-Dglucosaminidase (Rosado et al, 1977) and, less clearly, of alkaline phosphatase and arylsulphatase in saliva (Boyer and France, 1976). However, these salivary enzymes are probably derived from lyzed exfoliated cells, whereas lactoperoxidase is actively secreted from the salivary glands, and hence, has the highest activity (see Table I). Peroxidase may provide a suitable marker for ovulation, although any final conclusion about the most sensitive enzyme marker must involve consideration of the relative magnitudes of the change at ovulation for all the above enzymes. However, peroxidase activity is affected by a number of factors, which may lead to poor
78 1
TABLEI A comparison of enzyme activities in the saliva from four women*
Enzyme
Peroxidase N-acetyl-,%D glucosaminidase Acid phosphatase Alkaline phosphatase ,8-Glucuronidase Sulphatase A+B
Activity (pmol min-' g'lprotein) MeankSEM
Factor for conversion of activity to absorbance (change min-' g-' protein)
271.0 *46.3
x 32.4
10.60 0.79 8.90 f 0.96
x 17.8
8.75 f 1.01 0.101 f 0.029 0.358* 0.093
x 17.8 x17.8 x29.6 x 4.6
* The factor shown in the column to the right represents the micromolar extinction coefficient for the substrate used in each assay, which indicates the sensitivity of that substrate. Assays are described in materials and methods.
resolution of the mid-cycle peak. Lactoperoxidase is a 'haem' enzyme and is, therefore, very sensitive to changes in the oxidation state of the haem moiety. A number of foodstuffs appear to affect its activity as does the time of day (Fig. 3). Immunization (Fig. l), allergies (Fig. 5), and infection (Fig. 5) appear to stimulate peroxidase activity. There is a need for an easily applied, noninvasive method of ovulation detection which could be applied under a variety of conditions in populations with a wide range of educational background. The estimation of peroxidase may provide the basis for such a method, but its sensitivity to other variables such as infection, eating and time of day may present serious limitations in the use of this and possibly other salivary enzymes. ACKNOWLEDGEMENTS We are grateful for statistical advice from D. G. Altman, Clinical Research Centre, and for the assistance of Northwick Park Hospital, Clinical Chemistry Laboratories in hormone determinations. REFERENCES Attstrorn, R., and Egelberg, J. (1970): Journal of Periodontal Research, 5, 48.
782
COCKLE AND HARKNESS
Bain, B. J., and England, J. M. (1975): British Medical Journal, 2,473. Borooah, J., Leaback, D. H., and Walker, P. G. (1961): Biochemical Journal, 78, 106. Boyer, K. G., and France, J. T. (1976): International Journal of Fertility, 21,43. Fishman, W. H., Kato, K., Antiss, C. L., and Green, S. (1967): Clinica chimica acta, 15,435. Fridovich, I. (1976): Free Radicals in Biology. Volume 1. Academic Press, New York, p 239. Gothefors, L., and Marklund, S. (1975): Infection and Immunity, 11, 1210. Hausamen, T . U., Helger, R., Rick, W., and Gross, W. (1967): Clinica chimica acfa, 15, 241. Hogg, D. M., and Jago, G. R. (1970): Biochemical Journal, 117, 779. Kern, R., Wildbrett, G., and Kiermeier, F. (1963): Zeitschrift fur Naturforschung (B), 18, 1082. Klebanoff, S. J. (1968): Journal of Bacteriology, 95,2131. Lehrer, R. I. (1971): Journal of Clinical Investigation, 50, 2498. Lucas, F. V., Carnes, V. M., Schmidt, H. J., Sipes, D. R., and Hall, D. G. (1964): American Journal of Obstetrics and Gynecology, 88, 965. Lyttle, C. R., and De Sombre, E. R. (1977): Nature, 268, 337.
Lyttle, C. R., and Jellinck, P. H. (1972): Biochemical Journal, 127,481. Maehly, A. C., and Chance, B. (1954): Methods of Biochemical Analysis, 1, 357. Makinen, K. K., and Tenovuo, J. (1976): Acta odontologia Scandinavica, 34, 141. Miller, G. L. (1959): AnaZyticaZ Chemistry, 31, 964. Mitchell, R. H., Karnovsky, M. J., and Karnovsky, M. L. (1970): Biochemical Journal, 116,207. Paul, K . G. (1963): The Enzymes. Second edition, Volume 8. Academic Press, London and New York, p 227. Polishuk, W. Z., Diamant, Y. Z., Zuckerman, H., and Sadovsky, E. (1970): American Journal of Obstetrics and Gynecology, 107, 604. Rosado, A., Delgado, N. M., VelAzquez, A., Aznar, R., and Martiriez-Manautou, J. (1977): American Journal of Obstetrics and Gynecology, 128, 560. Rosner, F., and Lee, S. L. (1965): Blood, 25, 356. Shindler, J. S., Childs, R. E., and Bardsley, W. G. (1976): European Journal of Biochemistry, 65, 325. Thomson, J., and Morell, D. B. (1967): Journal of Biochemistry, 62, 483. Woollen, J. W., Heyworth, R., and Walker, P. G. (1961): Biochemical Journal, 78, 1 1 1 .
CHANGES IN SALIVARY PEROXIDASE AND POLYMORPHONUCLEAR NEUTROPHIL LEUCOCYTE ENZYME ACTIVITIES DURING THE MENSTRUAL CYCLE BY
S. M. COCKLE AND
R. A. HARKNESS Division of Perinatal Medicine, Clinical Research Centre Watford Road, Harrow HA1 3UJ, UK
Summary An elevation in salivary peroxidase activity has been found about the time of ovulation in 14 menstrual cycles in a total of six women. This peak coincided with the ovulatory luteinizing hormone (LH) and oestrogen peak in the four cycles in which endocrine studies were performed. Rises in some polymorphonuclear neutrophil leucocyte enzymes were also seen around ovulation. The possible use of changes in salivary peroxidase as a method for detection of ovulation is discussed.
in the human endometrium (Lucas et al, 1964) and peroxidase in bovine milk (Kern et al, 1963) show elevated activities about the time of ovulation. A fall in human milk peroxidase activity has been seen after delivery (Gothefors and Marklund, 1975) which may follow closely the rapid oestrogen withdrawal over the same period. Finally, physiological doses of oestrogen cause stimulation of uterine peroxidase activity in rats (Lyttle and Jellinck, 1972). We decided to investigate this effect of oestrogen using lactoperoxidase in human saliva as a model system, and report here the activities of peroxidase in salivary supernatant during the menstrual cycle in women. A second peroxidase, namely myeloperoxidase, was also studied about the time of ovulation, and changes compared with those found in salivary peroxidase. General enzymic effects were followed by studying other neutrophil enzymes.
PEROXIDASES are widespread and exist in a variety of molecular forms (Paul, 1963). They provide one of the pathways inside the cell for the breakdown of hydrogen peroxide, a reactive product of aerobic respiration (Fridovich, 1976), during the oxidation of a diverse range of substrates (Maehly and Chance, 1954). The distribution of peroxidases throughout the body fluids and tissues is highly variable (Lyttle and De Sombre, 1977), suggesting other physiological functions. For example, myeloperoxidase, in the polymorphonuclear neutrophil leucocyte (neutrophil) and lactoperoxidase found in saliva, milk and tears have bactericidal properties (Klebanoff, 1968; Hogg and Jago, 1970). Peroxidases may, therefore, play an important role in defence mechanisms against bacterial invasion. Investigations have indicated that oestrogens effect peroxidase production. Uterine peroxidase 776
SALIVARY PEROXIDASE DURING CYCLE
METHODS Clinical methods Volunteers (seven women and four men) collected 1 ml of saliva, before breakfast, in a universal container. The secretion of saliva and its composition vary considerably in a response to a variety of physiological circumstances, and because of this, the importance of collecting the samples a t the same time of day, before eating, and from the same part of the mouth was stressed. Samples, stored at 4 "C for up to five days, were centrifuged at 1500 g for 20 minutes and the supernatant retained for peroxidase and protein determinations. Salivary peroxidase activity is expressed relative to protein in preference to volume, as the water content of saliva varies considerably. Salivary peroxidase is stable for at least one week at 4 "C. A 20 ml sample of heparinized venous blood was collected daily from women at about the time of ovulation for neutrophil studies and estimation of plasma hormone levels. Approval for blood samples was obtained from the Brent and Harrow Health Authority Ethical Committee. Biochemical methods All materials were of analytical quality or of the highest purity available. 2,T-Azino di (3-ethyl benzothiazoline-6-sulphonicacid) ABTS, was obtained from Boehringer (Mannheim, Germany), and 6 per cent Dextran of molecular weight 110 000 in 0.9 per cent saline (Dextraven) from Fisons Ltd (Loughborough, Leicestershire, England). All other substrates, enzymes and reagents were obtained from Sigma Chemical Co (London, England). Peroxidase assay The assay was performed, using the chromogen ABTS, as described by Shindler et a1 (1976) with the modifications that the reaction was carried out at 25 "C in a final volume of 3 ml. The assay medium contained 1 mM ABTS and 0.1 mM hydrogen peroxide in 0.1 M sodium acetate buffer pH 4.4. The absorbence change was followed at 412 nm in a cuvette of 1 cm path length using a Unicam SP 1800 Spectrophotometer. The oxidation of one pmol of ABTS under the conditions of the assay
777
leads to a change in absorbence of 32.4. Units for all enzyme activities in this report are expressed as pmol of substrate utilized/ minute per g of protein. Total proteins were measured by the method of Miller (1959) using bovine serum albumin as the standard protein. Preparation of blood samples from women at about the time of ovulation A 20 ml sample of heparinized venous blood, collected daily around the time of ovulation, was centrifuged at 2000 g for five minutes. The blood cells were resuspended in isotonic saline and the neutrophils separated by a modification of the method of Lehrer (1971). Six per cent dextran in 0 - 9 per cent saline was added in the ratio three volumes dextran to ten volumes red cell suspension, and the erythrocytes allowed to sediment for 100 minutes at Ig. The neutrophil-rich supernatant was centrifuged at 2000 g for five minutes and the pellet retained. The cells were treated with 0.87 per cent (w/v) NH4CI to cause the lysis of contaminating erythrocytes, and then washed repeatedly in isotonic glucose until free from haemoglobin and red cell ghosts. The cells were resuspended in 1 ml of isotonic glucose, lyzed by freezing and thawing three times, and finally assayed for Pperoxidase, alkaline phosphatase, glucuronidase, acid phosphatase and total protein. Enzyme assays in neutrophils and saliva Alkaline phosphatase was assayed by the method of Hausamen et a1 (1967); acid phosphatase by the method of Mitchell et a1 (1970); and P-glucuronidase by the method of Fishman et a1 (1967). Arylsulphatase was assayed after removal of inhibitory phosphate ions from the saliva, according to the method of Boyer and France (1976). N-acetyl-P-D-glucosaminidase in saliva was assayed using a method based on those described by Borooah et al (1961) and Woollen et a1 (1961). The assay medium, of final volume 1 ml, contained 3 - 6 mM p-nitropheny1-Nacetyl-8-D-glucosaminide in 0.05 M citrate buffer pH 4.5, and an aliquot of saliva to
778
COCKLE AND HARKNESS
initiate the reaction. After incubation for 30 minutes at 37 “C, the reaction was stopped by addition of 2 ml of 0 . 2 M borate buffer (pH 9.8) and the absorbance at 405 nm was read against a suitable blank.
z.-
-
400 ;cul]
-
.-k
100
E E,
x
3
5
7
9
11 13 15 17 19 21 23 25 1
1 3
5
7
9
11 13 15 17 19 21 23 1 Day of cycle
1
0
RESULTS Salivary peroxidase activities Serial peroxidase activities in the saliva of seven women (ages 18 to 45) were studied throughout 15 menstrual cycles. All showed mid-cycle peaks in activity, with the exception of one from whom samples were not available over the ovulatory period. Ninety-nine per cent confidence limits for peroxidase activity were calculated for all cycles. Eight consecutive activities, during either the early follicular or late luteal phases, were used to calculate the mean and its confidence limits. With the exception of one woman, B, the mid-cycle peaks of peroxidase activity lay outside these confidence limits. Peroxidase activities throughout five complete menstrual cycles are shown in Figures 1,2 and 3. The magnitude of the mid-cycle peak is variable, showing about a two-fold rise in three of these cycles (subject A) and about a three-fold rise in subjects C and D. The peak is considerably smaller in subject B (Fig. 6 ) . Two consecutive cycles from one woman, A, show repeatable patterns of peroxidase activity throughout
7s”^.ryl;,
300
ze
200
I?
100
-
FIG.2 Salivary peroxidase activities throughout two consecutive menstrual cycles in one woman, A. The solid bars represent menstruation.
1 4 5 0 1 Subject C
E
50
3
5
7
9 11 13 15 17 19 21 23 2 Day of cycle
4
FIG.3 Salivary peroxidase activities during one cycle in one woman, A. Both early morning (0730) and late evening (2300) saliva samples were studied. Total oestrogen excretion per 24 hours is shown over the ovulatory period. The solid bars represent menstruation.
;350
.-
3
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
15 17 19 21 23 25 27 29 31 33 35 37 39 41 431 3 500
Subject
D
f bysler) immun.
._
2
P
200
100 1
3
5
7
9 11 13 15 17 19 21 23 25 2 4 Day of cycle
FIG.1 Salivary peroxidase activities during one menstrual cycle in two women, C and D. The solid bars represent menstruation.
the cycle, as well as clear peaks at mid-cycle. The results in Figure 3, also from subject A, show that serial activities at 0730 and 2300 hours both have a clear increase at mid-cycle but all activities are about two to three times higher in the evening. The results from 14 cycles are superimposed as a scatter diagram (Fig. 4). The cycle in which data were not available over the ovulatory period was omitted. The time-scale refers to the number of days before menstruation
SALIVARY PEROXIDASE DURING CYCLE
779
- -
u
Cold
Subject H
600
n
1
26 24 22 20 18 16 14 12 10 8 6 4 Time in days before last menstruation
2
FIG.4 Accumulated peroxidase activities from 14 menstrual cycles. Peroxidase activity is expressed as a multiple of a mean value which is calculated from eight consecutive values from either the early follicular or late luteal phases of the cycle.
because the luteal phase tends to be more constant than the follicular phase of the menstrual cycle. Most points lie within a broad band of activity but over a mid-cycle period (days 11 to 19) eight points lie clear of this band with activities two to three times the mean. Serial peroxidase activities in the saliva of four men were studied throughout one month (Fig. 5). In contrast to the results for women, there is no common pattern. Peroxidase activities, in subjects E and F, vary only slightly throughout the month while G and H show marked elevations on certain days. After primary immunization, against tetanus toxoid and poliovirus of subject C (Fig. 1) and of a second woman B (not illustrated), there was a distinct elevation of salivary peroxidase activity over five days, followed by a rapid fall to previous levels. Subject D who only received a 'booster' dose of tetanus toxoid and poliovirus did not show this elevation. In the man, G, an allergic reaction to animals coincided with an elevation of salivary peroxidase activity, and an upper respiratory tract infection, in H, also appeared to elevate the activity.
3
5
7
9
11 13 I5 17 19 21 23 25 27 Day of month
FIG.5 Salivary peroxidase activities throughout one month in four men.
Relation to ovulation Endocrine data have confirmed that the mid-cycle peak of peroxidase coincides with elevations of plasma oestradiol, determined by radioimmunoassay, in three cycles (Figs. 6 , 7 and 8) and of total urinary oestrogens, determined fluorimetrically, in one cycle (Fig. 3). The plasma luteinizing hormone (LH) peak, determined by radioimmunoassay, occurs within one day of the peroxidase peak. Elevations in urinary pregnanediol, determined by gas liquid chromatography, after the peroxidase peak, confirmed the presence of a functioning corpus luteum in subjects A and B. Polymorphonuclear neutrophil Ieucocyte enzyme activities A selection of neutrophil enzyme activities were studied over the ovulatory period in three cycles (Figs. 6 , 7 and 8). In general, the patterns of enzyme activities in neutrophils are less clear than those for saliva as it was not practicable to obtain data throughout the whole month. However, elevations in neutrophil peroxidase, alkaline phosphatase and P-glucuronidase activities do appear to occur concurrently with rises in plasma oestradiol-17P in two women (Figs. 6 , 7 and 8). DISCUSSION A mid-cycle peak in salivary peroxidase which coincides with the ovulatory oestrogen
780
COCKLE AND HARKNESS I
2000
1 Cycle 3
I
I
Plasma Oestradiol
500
- 17 p
-
500
15w
' '"1
I
Leuc. Peroxidase
+ y c .
Alk. Phos.
:::1 0.9
0
- 3 - 2 - 1 0
1
2
3
4
Days before a n d after LH peak
FIG.6 Salivary peroxidase and neutrophil peroxidase and alkaline phosphatase activities around the time of ovulation in subject B (cycle 3). Enzyme activities and plasma oestradiol-l7,G are plotted relative to the LH peak.
Plasma Oeslradiol-176
Saliva Peroxidase
j
'"1 +
-Glucuronidase
'.j & 0
2w 1
Leuc. Alk. Phos
0
-5 -4 - 3 -2 -1 0 1 2 3 4 Days before and after LH peak
FIG.7 Salivary peroxidase and neutrophil peroxidase, ,G-glucuronidase and alkaline phosphatase activities around the time of ovulation in subject B (cycle 4). Enzyme activities and plasma oestradiol-179 are plotted relative to the LH peak.
w - G l u c u r o n i d a s e
,,
-id
Phosphatase
0 -3 - 2 -1 0 1 2 3 Days before and after LH peak
FIG.8 Salivary peroxidase and neutrophil peroxidase, alkaline phosphatase, p-glucuronidase and acid phosphatase activities around the time of ovulation in subject A (cycle 3). Enzyme activities and plasma oestradiol-l7,G are plotted relative to the LH peak.
peak has been found in all women from whom samples were available over the ovulatory period. The effects were repeatable in consecutive cycles in the same woman, and the peaks were of comparable magnitude, although their size varied markedly in different women. There was a marked contrast between the individual plots, and the scatter plot representation (Fig. 4). This was because of large variation between women. A peak shown clearly by an individual may be no higher than the generality of non-peak values shown by other women. Also, in the scatter plot a large variation in time of ovulation led to poor peak definition. Lactoperoxidase is possibly responsible for the change in salivary peroxidase activity seen at ovulation. It is the main peroxidase present in saliva, and is actively secreted from the salivary glands (Thomson and Morell, 1967). Myeloperoxidase, from neutrophils which migrate through the gingival crevice (Attstrom and Egelberg, 1970) and bacterial peroxidase are also present in saliva. Centrifugation of the saliva largely removed these cellular contaminants, although low concentrations of
SALIVARY PEROXIDASE DURING CYCLE
both peroxidases may be present in the supernatant due to lysis of the cells (Makinen and Tenovuo, 1976). The mid-cycle elevations in neutrophil enzyme activities, demonstrated here, are more difficult to interpret than those for salivary peroxidase. All activities are expressed per mg of total protein, and hence, are independent of the total number of cells in our preparation. It has been demonstrated that circulating neutrophil numbers increase around the time of ovulation in women (Bain and England, 1975), so that the elevations of neutrophil enzyme activities could be reflecting a proportional increase in neutrophil numbers within our white cell preparation. This cannot be the case as the preparation of cells used consisted of about 95 per cent neutrophils. Peaks in neutrophil alkaline phosphatase activities were found in this study, which are consistent with previous evidence for oestrogenic control of neutrophil alkaline phosphatase. For example, its activity is raised two to threefold during pregnancy and follows changes in oestrogen production (Polishuk et al, 1970). In addition, women aged between 19 and 48 have significantly higher neutrophil alkaline phosphatase activities than men, and the activities fall after the menopause (Rosner and Lee, 1965). The mid-cycle peak in salivary peroxidase activity could perhaps be used to detect ovulation. Saliva is a convenient and abundant source of peroxidase, and the assay is quick and sensitive (Table I). Previous workers have found peaks of N-acetyl-p-Dglucosaminidase (Rosado et al, 1977) and, less clearly, of alkaline phosphatase and arylsulphatase in saliva (Boyer and France, 1976). However, these salivary enzymes are probably derived from lyzed exfoliated cells, whereas lactoperoxidase is actively secreted from the salivary glands, and hence, has the highest activity (see Table I). Peroxidase may provide a suitable marker for ovulation, although any final conclusion about the most sensitive enzyme marker must involve consideration of the relative magnitudes of the change at ovulation for all the above enzymes. However, peroxidase activity is affected by a number of factors, which may lead to poor
78 1
TABLEI A comparison of enzyme activities in the saliva from four women*
Enzyme
Peroxidase N-acetyl-,%D glucosaminidase Acid phosphatase Alkaline phosphatase ,8-Glucuronidase Sulphatase A+B
Activity (pmol min-' g'lprotein) MeankSEM
Factor for conversion of activity to absorbance (change min-' g-' protein)
271.0 *46.3
x 32.4
10.60 0.79 8.90 f 0.96
x 17.8
8.75 f 1.01 0.101 f 0.029 0.358* 0.093
x 17.8 x17.8 x29.6 x 4.6
* The factor shown in the column to the right represents the micromolar extinction coefficient for the substrate used in each assay, which indicates the sensitivity of that substrate. Assays are described in materials and methods.
resolution of the mid-cycle peak. Lactoperoxidase is a 'haem' enzyme and is, therefore, very sensitive to changes in the oxidation state of the haem moiety. A number of foodstuffs appear to affect its activity as does the time of day (Fig. 3). Immunization (Fig. l), allergies (Fig. 5), and infection (Fig. 5) appear to stimulate peroxidase activity. There is a need for an easily applied, noninvasive method of ovulation detection which could be applied under a variety of conditions in populations with a wide range of educational background. The estimation of peroxidase may provide the basis for such a method, but its sensitivity to other variables such as infection, eating and time of day may present serious limitations in the use of this and possibly other salivary enzymes. ACKNOWLEDGEMENTS We are grateful for statistical advice from D. G. Altman, Clinical Research Centre, and for the assistance of Northwick Park Hospital, Clinical Chemistry Laboratories in hormone determinations. REFERENCES Attstrorn, R., and Egelberg, J. (1970): Journal of Periodontal Research, 5, 48.
782
COCKLE AND HARKNESS
Bain, B. J., and England, J. M. (1975): British Medical Journal, 2,473. Borooah, J., Leaback, D. H., and Walker, P. G. (1961): Biochemical Journal, 78, 106. Boyer, K. G., and France, J. T. (1976): International Journal of Fertility, 21,43. Fishman, W. H., Kato, K., Antiss, C. L., and Green, S. (1967): Clinica chimica acta, 15,435. Fridovich, I. (1976): Free Radicals in Biology. Volume 1. Academic Press, New York, p 239. Gothefors, L., and Marklund, S. (1975): Infection and Immunity, 11, 1210. Hausamen, T . U., Helger, R., Rick, W., and Gross, W. (1967): Clinica chimica acfa, 15, 241. Hogg, D. M., and Jago, G. R. (1970): Biochemical Journal, 117, 779. Kern, R., Wildbrett, G., and Kiermeier, F. (1963): Zeitschrift fur Naturforschung (B), 18, 1082. Klebanoff, S. J. (1968): Journal of Bacteriology, 95,2131. Lehrer, R. I. (1971): Journal of Clinical Investigation, 50, 2498. Lucas, F. V., Carnes, V. M., Schmidt, H. J., Sipes, D. R., and Hall, D. G. (1964): American Journal of Obstetrics and Gynecology, 88, 965. Lyttle, C. R., and De Sombre, E. R. (1977): Nature, 268, 337.
Lyttle, C. R., and Jellinck, P. H. (1972): Biochemical Journal, 127,481. Maehly, A. C., and Chance, B. (1954): Methods of Biochemical Analysis, 1, 357. Makinen, K. K., and Tenovuo, J. (1976): Acta odontologia Scandinavica, 34, 141. Miller, G. L. (1959): AnaZyticaZ Chemistry, 31, 964. Mitchell, R. H., Karnovsky, M. J., and Karnovsky, M. L. (1970): Biochemical Journal, 116,207. Paul, K . G. (1963): The Enzymes. Second edition, Volume 8. Academic Press, London and New York, p 227. Polishuk, W. Z., Diamant, Y. Z., Zuckerman, H., and Sadovsky, E. (1970): American Journal of Obstetrics and Gynecology, 107, 604. Rosado, A., Delgado, N. M., VelAzquez, A., Aznar, R., and Martiriez-Manautou, J. (1977): American Journal of Obstetrics and Gynecology, 128, 560. Rosner, F., and Lee, S. L. (1965): Blood, 25, 356. Shindler, J. S., Childs, R. E., and Bardsley, W. G. (1976): European Journal of Biochemistry, 65, 325. Thomson, J., and Morell, D. B. (1967): Journal of Biochemistry, 62, 483. Woollen, J. W., Heyworth, R., and Walker, P. G. (1961): Biochemical Journal, 78, 1 1 1 .
