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Evaluation of leukocyte arylsulfatase-A activity in patients with breast cancer and benign breast disease
Cancer Letters 166 (2001) 95±101
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Evaluation of leukocyte arylsulfatase-A activity in patients with breast cancer and benign breast disease È ner b,*, FuÈsun CËõnar b, Hikmet KocËak b, GuÈvencË GuÈvenen c, Sembol TuÈrkmen a, Pernur O HuÈseyin Altun d, Yavuz Eryavuz e a Department of Biochemistry, Social Insurance Institution, Okmeydanõ Educational Hospital, Istanbul, Turkey Department of Biochemistry, Istanbul Faculty of Medicine, University of Istanbul, CËapa 34390, Istanbul, Turkey c Department of Biochemistry, Social Insurance Institution, Istanbul Educational Hospital, Istanbul, Turkey d Department of General Surgery, Social Insurance Institution, Istanbul Educational Hospital, Istanbul, Turkey e Department of General Surgery, Social Insurance Institution, Okmeydanõ Educational Hospital, Istanbul, Turkey b
Received 15 November 2000; received in revised form 25 January 2001; accepted 29 January 2001
Abstract This study was planned to evaluate the feasibility of using the assay of leukocyte arylsulfatase-A (AS-A) activity as a noninvasive diagnostic tool in patients with benign and malignant breast disease. The leukocyte AS-A activity of a total of 81 women was analyzed, including 28 healthy women, 29 women with benign breast disease (BBD) and 24 patients with primary breast cancer (BC). The mean leukocyte AS-A activity in patients with BBD was slightly higher (14.3%) that observed in the healthy subjects, but the difference was not statistically signi®cant. In patients with BC the enzyme activity was signi®cantly higher than in the healthy subjects (60.3%, P , 0:05) and in the benign group (40.2%, P , 0:05). In addition, since no signi®cant differences have been observed between premenopausal patients and their controls, it is suggested that the measurement of leukocyte AS-A activity may not be a reliable test for differential diagnosis of benign and malignant proliferation in mammary glands due to the possible interfering effect of gonadal hormones on AS-A activity. In contrast, since peri- and postmenopausal BC patients have negligible or no gonadal activity function, the elevation in the activity of leukocyte AS-A in these age groups of patients may only be expected to originate from malignant proliferation. Based on our results, it is concluded that in patients in whom high leukocyte AS-A activities were observed the possibility of the presence of malignancy might also be high. Therefore, this test might be valuable as a non-invasive biochemical technique in combination with other established markers for the identi®cation of masses in the breast. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Leukocyte arylsulfatase-A; Benign breast disease; Breast cancer
1. Introduction Metabolic and especially anabolic processes are intensi®ed in the neoplasm and this manifests itself as a changed activity of a number of enzymes. More * Corresponding author. Tel.: 190-212-6311323-120; fax: 190212-6311323-115. È ner). E-mail address: [email protected] (P. O
than four decades ago, it was ®rst suggested that increased activity of arylsulfatases (ASs E.C.3.1.6.1.), which catalyze the hydrolysis of aromatic sulfate esters in malignant tissues [1] and also in the urine of patients with various active cancer [2,3], might be of diagnostic value. Since then, biochemical enzyme analyses have attracted great attention for better understanding of the biological
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00429-3
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S. TuÈrkmen et al. / Cancer Letters 166 (2001) 95±101
nature of malignancies. Results of some previous studies indicated that measurement of arylsulfataseA (AS-A), a primary lysosomal enzyme, might be useful in the diagnosis and/or monitoring of the malignancy [4±10]. On the other hand, breast cancer (BC) is now recognized as probably the most prevalent malignant transformation occurring in women. In order to investigate the diagnosis and characterization of masses in the body with the aid of non-invasive biochemical techniques, BC is used as a best representative model [11]. There are limited reports concerning the activity of leukocyte AS-A in patients with several malignant tumors, but no such data in patients with BC are available to date. In the light of these data, this study was planned to investigate to what extent leukocyte AS-A activity could serve as an appropriate and reliable non-invasive diagnostic tool for malignant and benign cell proliferation in patients with newly diagnosed BC and benign breast disease (BBD), and also to provide some contribution to very limited data on this subject. 2. Materials and methods A total of 81 women participated in this study. The patient population consisted of 53 women who were admitted to the Department of General Surgery of the Istanbul Educational Hospital of the Social Insurance Institution because of pain or a palpable nodule in their mammary tissue. Based on their histopathology and/or ultrasonographic or mammographic examination, 29 of 53 patients ranging in age from 20 to 59 years were grouped as BBD and the remaining patients with ages ranging from 31 to 67 years were grouped as BC. Twenty-three of the BBD patients had ®brocystic disease, three had ®broadenoma, one had adenosis, one had lipomatosis and one had both lobular granulomatous mastitis and ®brocystic disease. Of the 24 newly diagnosed, untreated BC patients, 17 had only invasive ductal carcinoma, two had invasive ductal carcinoma with tubular differentiation, three had only lobular carcinoma, one had invasive ductal carcinoma with osteoclastic giant cells and one had only mucinous carcinoma. As a control group, 28 healthy women with ages ranging from 25 to 65 years and with no evidence of any type of cancer or
in¯ammatory disease were studied. None of the study population had hormonal or drug therapy including vitamin supplementation. Subjects with a history of an allergic or anaphylactic reaction and pregnant or lactating women were excluded from the study. The participants were not restricted to a speci®c diet. Informed consent was obtained from each participant. Menopausal status at diagnosis was not speci®ed by hormone analysis, but women with ages ranging from 20 to 37 years who had regular menstrual cycles were labeled as premenopausal. Postmenopausal women were de®ned as those who had no menstrual bleeding in the 3 years prior to study and women with ages ranging from 38 to 50 years were designated as perimenopausal. Blood samples from patients with BC or BBD were obtained prior to any surgical or other therapeutic procedures. Fasting venous blood (11 ml; 10 ml of it anticoagulated with Heparin (Liquemine, Roche) and the remainder with EDTA) was drawn from each subject. Leukocytes were prepared from whole blood by adding an equal volume of Dextran (Sigma) in 0.85% saline and AS-A activity in leukocytes was measured using p-nitrocathecolsulphate (Sigma) as a substrate by the method of Pilz [12]. The amount of liberated nitrocathecol (NC) was determined by absorption at 515 nm in a LKB ultraspec 4050 spectrophotometer. Enzyme activity was expressed as nanomoles of hydrolyzed p-nitrocathecol per milligram of protein per hour at 378C. Protein was determined by the Lowry procedure [13] using serum albumin as the standard. All assays were run in duplicate and the results were the mean of duplicate measurements. The results for BC patients were compared with the results for healthy subjects and patients with BBD. Venous blood (1 ml) anticoagulated with EDTA was used for white blood cell counts using a Coulter JS counter. Comparisons between the healthy subjects and patients were performed using one-way ANOVA followed by a post-hoc Tukey HSD test and probability values of 0.05 or less were considered signi®cant. Differences in leukocyte AS-A activity between the age groups were tested using the Kruskal±Wallis test. The software used for all statistical evaluations was the SPSS 7.5 statistical package program. To estimate the validity of AS-A, sensitivity, speci®city and predictive values of the test were calculated.
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Table 1 Leukocyte AS-A activity in patients with benign and malignant breast disease and in healthy women (mean ^ SD)
Leukocyte AS-A (nmol NC/mg protein per h) a
Healthy women (n 28)
BBD patients (n 29)
BC patients (n 24)
83.6 ^ 16.8
95.6 ^ 27.6
134.0 ^ 52.0 a
P , 0:05 compared to control and BBD groups (Tukey HSD multiple comparison test).
The cut-off level for AS-A was established as 127.0 nmol NC based on leukocyte levels excluding approximately 97% of the healthy controls (this level is approximately equivalent to the mean 1 2.5 SD derived from the data). 3. Results Leukocyte AS-A, activities for normal subjects, patients with histologically con®rmed BC and patients with BBD are shown in Table 1 and Fig. 1. Leukocyte AS-A activities in healthy women, and in patients with BBD and BC ranged from 64.0 to 127.0 (mean 83.6 ^ 16.8), from 51.0 to 148.0 (mean 95.6 ^ 27.6), and from 68.0 to 256 (mean 134.0 ^ 52.0) nmol NC/ mg protein per h, respectively. Although the lower and upper enzyme activity levels in BBD and BC
Fig. 1. Distribution of leukocyte AS-A activity in normal women and patients with BBD and BC and values of the main statistical parameters.
patients were more widely distributed, the mean values observed in both non-malignant and malignant groups were found to be approximately 15 and 60% higher, respectively, than those of the control group. The difference reached statistical signi®cance in BC patients (P , 0:05, Table 1). In addition, leukocyte AS-A activity in patients with BC was 40.2% higher that observed in BBD patients, being the difference at the level of P , 0:05 (Table 1). The groups were divided into three subgroups according to their gonadal activity status as: premenopausal, aged 20±37 years (n 20); perimenopausal, aged 38±50 years (n 30); and postmenopausal, aged 51±67 years (n 31). The mean leukocyte ASA activities in patients aged 20±37 years with benign and malignant breast disease were not signi®cantly different from those observed in aged-matched controls (Table 2). In patients with BBD and BC aged between 38 and 50 years, the mean levels of leukocyte AS-A activity were found to be elevated compared with agedmatched controls, but only the difference between the levels of AS-A activity for BC patients and the control group was statistically signi®cant at a borderline level (P 0:056). The difference between the groups did not exhibit a high degree of signi®cance due to the small number of cases in both groups. The highest leukocyte AS-A activity was found in BC patients aged 51±67 years as compared to BC patients in pre- and perimenopausal categories. The mean leukocyte AS-A activity in postmenopausal BC patients aged 51±67 years was signi®cantly higher than those of aged-matched controls and BBD patients (P 0:001) (Table 2). The enzyme activity observed in postmenopausal BBD patients was found to be slightly but signi®cantly lower (P 0:001) than the aged-matched control group. When we used the upper limit of the normal range as 127 nmol NC/mg protein per h for leukocyte AS-A,
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Table 2 Leukocyte AS-A activities (nmol NC/mg protein per h) in patients and in healthy women at different ages (mean ^ SD) a Age ranges (years)
Healthy women
BBD patients
BC patients
20±37 38±50 51±67
111.0 ^ 15.8 (5) 79.5 ^ 12.0 (7) 75.6 ^ 9.8 (16)
99.8 ^ 22.1 (11) 98.9 ^ 30.4 (15) 63.6 ^ 6.6 (3)*
94.2 ^ 20.9 (4) 127.8 ^ 42.4 (8) 151.5 ^ 58.8 (12)*
a
The number of subjects is in parentheses. *P 0:001 compared to control and BBD groups.
inconclusive because of the lower level of the enzyme in blood than in urine [6,14,15]. Nevertheless, human neutrophil leukocytes are rich sources of ASs and peripheral circulating leukocytes often provide a convenient nucleated cell model for studying the biochemistry and pathology of other nucleated cells in the body [16,17]. Therefore, we preferably investigated the AS-A activity in peripheral leukocytes obtained from newly diagnosed, untreated BC patients and from BBD patients. The upper and lower levels of leukocyte AS-A activity in the method used by us are reported as 126.0 and 52.4 nmol NC/mg protein per h, respectively, with a mean value of 72.3 nmol NC [12]. Thus, our values observed for healthy subjects and for the majority of BBD patients (86.3%) are well within the reported values of Pilz [12] and those of other investigators [9,12,18±20]. The mean leukocyte AS-A activity value in patients with BBD did not differ signi®cantly from controls. This ®nding is similar to that obtained in a previous study reporting that AS-A activity in ®brocystic tissue did not differ distinctly from that found in normal tissue [4]. Our ®nding of a non-signi®cant difference between the mean value of leukocyte AS-A for normal subjects and for BBD patients suggested that the measurement of leukocyte AS-A may not be valuable for the diag-
the activity of 1/28 (3.5%) healthy women, 4/29 (13.7%) patients with BBD and 12/24 (50%) patients with BC was at or above this cut-off level. The sensitivity, speci®city and predictive values of a positive result were 0.50, 0.96, and 0.92, respectively, at the 127 nmol NC cut-off level. The overall test accuracy was 48.1% (Table 3). 4. Discussion In recent years, there has been expanding interest in the use of normal antigens, hormones, and enzymes for the diagnosis and prognosis of tumors and also for the assessment of treatment response. However, very few published clinical studies of the value of leukocyte AS-A activity in BC patients are available to date. In one of the preliminary studies performed on a very small number of BC patients, the activity of ASA was found to be signi®cantly increased in mammary tissues of three of ®ve BC patients examined, but in two cases pretreated with X-ray and in two BBD patients the level of the enzyme was found to be normal [4]. Analysis of the blood level of AS-A in several pathological states is scanty and results are generally
Table 3 Sensitivity, speci®city and predictive values of the leukocyte AS-A activity a Sensitivity
A B a
0.50 0.62
Speci®city
0.96 0.82
Predictive values
Overall test accuracy
(1)
(2)
0.92 0.75
0.69 0.60
0.48 0.46
Values are shown at 127 nmol NC (A) and 100.4 nmol (B) cut-off levels. The cut-off level 100.4 nmol NC is derived from data based on the mean 1 1 SD.
S. TuÈrkmen et al. / Cancer Letters 166 (2001) 95±101
nosis of BBD. In contrast, the more pronounced activity of leukocyte AS-A in BC patients than in healthy subjects (60.3%) and BBD patients (40.2%) strengthened the conclusion that the measurement of enzyme activity may be useful in the differential diagnosis of benign and malignant proliferation. In addition, our ®ndings concerning the value of variation coef®cients for AS-A activity in controls and patients with BC is similar to the ®ndings of Laidler et al. [7], who also found a 25.5% variation coef®cient for the serum ASA concentration in patients with BC. To the best of our knowledge, this is the ®rst report concerning the measurement of leukocyte AS-A activity both in patients with BC and with BBD. Consequently, there are no available data for direct comparison with our results. However, highly increased leukocyte AS-A activities in leukemia patients have been previously reported [9,21]. High leukocyte AS-A activity in BC patients observed by us suggested that patients having high AS-A activity in leukocytes and/or tumor tissues might be associated with malignant disease. The ®ndings of previous studies reporting elevated levels of AS-A in solid tumor tissues, leukocytes and the urine of patients with various cancers seem to support our suggestion [4,5,8,9,21± 23]. The activity of leukocyte AS-A in patients with BC was increased above the upper limit of the normal range (127 nmol NC) in 50% of cases. This result is comparable with the result obtained in an earlier work reporting that an elevated value for CA 15.3 over the upper cut-off level was also observed for 48.7% of BC patients before surgery [24]. However, on the basis of this ®nding it is concluded that the presence of the tumor should not be excluded when the tumor marker levels are below the cut-off point. Accordingly, because half of our BC patients had a value of leukocyte AS-A activity above the upper limit of the normal range, it should not be thought that measurement of leukocyte AS-A activity is not suf®ciently valuable for suspecting the presence of malignancy. In addition, 66% of the remaining BC patients were above the mean level of leukocyte AS-A activity for healthy women. Previous research on lysosomal enzymes including ASs and their possible connection with endocrine functions strongly suggest the in¯uence of hormonal factors on AS-A activity [23,25±29]. In one of these
99
studies, increased leukocyte AS-A activity in healthy premenopausal women during ovulatory and luteal phases was observed [28]. In the other studies, elevations in both AS-A and B activities in experimental animals during physiological estrus was observed [27,29]. The results of these observations suggest that elevations in leukocyte and urine AS-A activities may be observed without any pathological cause under physiological or experimental conditions, especially in hyperestrogenic subjects [28]. Furthermore, in a previous study performed on human endometrium in which proliferation and differentiation are controlled by sex hormones, the levels of AS-A in endometrium biopsy samples from women with cystic hyperplasia or adenocarcinoma were also found to be higher, being more pronounced in the neoplastic endometrium than those obtained from normal women in the proliferative phase [23]. On examination of the three subgroups which were divided according to their gonadal activity status as pre-, peri-, and postmenopausal groups, the mean values of leukocyte AS-A activities in premenopausal benign and malignant breast disease patients were not found to be signi®cantly different than those of agematched controls. This ®nding may be due to the randomness of the time of blood collection and perhaps it is at the time of the menstrual period that gonadal steroids are in¯uential. The reason for not standardizing the blood collection time was the obligation to obtain blood specimens from carcinoma patients prior to surgical intervention. Therefore, one might suggest that in women having in¯uential gonadal activity function, the measurement of leukocyte AS-A activity may not provide a reliable test for use in the differential diagnosis of benign and malignant proliferation in mammary glands. Since perimenopausal BC patients have little gonadal activity function and postmenopausal patients have absolutely no gonadal function, the elevations in the activity of leukocyte AS-A in BC patients between 38±50 and 51±67 years of age in our study may only be expected to originate from neoplastic proliferation. In addition, it should be of considerable interest to investigate ASA activity in BC patients treated with the antiestrogenic drug Tamoxifen. The lack of statistical signi®cance between perimenopausal BC and BBD patients aged between 38 and 50 years may be due to the smaller sample size and reduced statistical power.
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In our study, the highly signi®cant AS-A concentration in leukocytes of BC patients could have resulted from intensi®ed cell metabolism and/or stimulated synthesis of the enzyme due to a cytokine produced by malignant cells or their surrounding cells. In discriminating healthy cases from patients with BC using the AS-A cut-off limit value, only 1/28 healthy individuals were false positive and 12/24 BC patients were false negative, leading to a speci®city of 0.96 and a sensitivity of 0.50. Accordingly, the test seems to be able to detect 50% of BCs with a speci®city approaching 100%. In addition, our ®ndings of an overall spectrophotometric test accuracy of 48.1% and positive and negative predictive values of 92.3 and 69.2%, respectively, suggested that measurement of leukocyte AS-A might provide a helpful noninvasive test for the diagnostic work-up. From the results obtained in our study, it can be concluded that in patients having high leukocyte AS-A activity, the probability of the presence of malignancy might also be high, and therefore this test might be valuable as a non-invasive biochemical technique in combination with other established markers for the identi®cation of masses in the breast. Acknowledgements The authors would like to thank Drs Nurten Turan È mit TuÈrkogÏlu for their help in data analyses. and U References [1] E. Boyland, D.M. Wallace, D.C. Williams, The activity of the enzymes sulphatase and b-glucuronidase in the urine, serum and bladder, Br. J. Cancer 9 (1995) 62±67. [2] L.M. Dzialoszynski, The clinical value of arylsulfatase estimation in urine, Clin. Chim. Acta 2 (1957) 542±547. [3] L.E. Posey, L.R. Morgan, Measurement of arylsulphatase A activity in urine, Clin. Chem. 25 (1979) 328±331. [4] L.M. Dzialoszynski, J.L. Kroll, A. FroÈchlich, Arylsulphatase activity of some malignant tumors, Clin. Chim. Acta 14 (1966) 450±453. [5] S. Gasa, A. Makita, T. Kameya, T. Kodama, E. Araki, T. Yoneyama, M. Hirama, M. Hashimoto, Elevated activities and properties of arylsulfatase A and B variant in human lung tumors, Cancer Res. 40 (1980) 3804±3809. [6] K. Honke, T. Fujii, A. Makita, Arylsulfatase A protein in sera of cancer patients, Cancer J. 8 (1995) 139±142.
[7] P. Laidler, D. Kowalski, J. Silberring, Arylsulphatase A in serum from patients with cancer of various organs, Clin. Chim. Acta 204 (1991) 69±78. [8] K. Mitsuhashi, A. Maru, T. Kayanagi, T. Ishibashi, Y. Imai, S. Gasa, N. Taniguchi, A. Makita, Arylsulphatase A activities in urine and tissues taken from bladder cancer patients, Jpn. J. Exp. Med. 54 (1984) 211±216. È mer, P. O È ner, M. Manyaslõ, M. Aktan, Leukocyte aryl[9] B. O sulphatase A activity in acute leukemia, Cancer Biochem. Biophys. 14 (1994) 53±56. [10] L.E. Posey, J. Rainey, R. Vial, D. Ryan, J.H. Bickers, R.L. Morgan Jr., M.S. Samuels, E.W. Hull, A noninvasive technique for monitoring response to chemotherapy in human acute leukemia, Cancer 44 (1979) 873±880. [11] G.F. Schwartz, R. Schwarting, P. Rabindranauth, G.C. Finkel, Clinical applications of serum and tissue markers in malignant disease: breast cancer as a paradigm, Clin. Chem. 39 (11) (1993) 2404±2412. [12] H. Pilz, Late adult metachromatic leukodystrophy, Arch. Neurol. 27 (1972) 87±90. [13] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265±275. [14] A.J. Gniot-Szulzycka, Arylsulphatase A and B in blood, Clin. Chim. Acta 32 (1971) 17±23. [15] P.M. Laidler, K.W. Ryder, M.R. Glick, T.O. Oei, R.L. Van Etten, Radioimmunoassay for arylsulphatase A in urine, Clin. Chem. 31 (1985) 391±396. [16] M.C. Geokas, H. Rinderknecht, Plasma arylsulphatase and bglucoronidase in acute alcoholism, Clin. Chim. Acta 46 (1973) 27±32. [17] P. Martinelli, M. Montanari, M. Ippoliti, M. Mochi, S. Sangiorgi, M. Capocasa, Familial spasmodic dysphonia with low arylsulphatase A (AS-A) level, Acta Neurol. Scand. 91 (1995) 196±199. [18] B. Herz, G. Bach, Arylsulphatase A in pseudode®ciency, Hum. Genet. 66 (1984) 147±150. [19] M.J.A.J.M. Hoes, K.J.B. Lamers, R.A. Wevers, Use of arylsulphatase A in chronic psychiatric patients, Am. J. Psychiat. 145 (1988) 382. [20] P. Propping, W. Friedl, M. Huschka, K.H. SchloÈr, F. Reimer, M. Lee-Vaupel, E. Conzelmann, K. Sandhoff, The in¯uence of low arylsulphatase activity on neuropsychiatric morbidity: a large-scale screening in patients, Hum. Genet. 74 (1986) 244±248. [21] Y. Uehara, S. Gasa, A. Makita, K. Sakurada, T. Kiyazaki, Lysosomal arylsulfatase of human leukocytes. Increment of phosphorylated B variants in chronic myelogenous leukemia, Cancer Res. 43 (1983) 5618±5622. [22] N. Ueno, T. Matsumoto, M. Ohkawa, M. Nakayama, T. Tonoka, Urinary arylsulphatase in normal children and in patients with pediatric malignant disease, Tohoku J. Exp. Med. 139 (1983) 165±169. [23] L. Vitaioli, E. Baldoni, R. Ricci, S.R. Indraccolo, Expression of acidic glycosphingolipids and arylsulphatase A activity in human pathological endometrium, Eur. J. Obstet. Gynecol. Reprod. Biol. 54 (1994) 31±35.
S. TuÈrkmen et al. / Cancer Letters 166 (2001) 95±101 [24] M. Gion, G. Ruggeri, R. Marconato, C. Casella, A. Nosadini, G. Laffranchini, R. Mione, S. Belloli, G. Bruscagnin, Perioperatory kinetic evaluation of tumor markers in breast and colorectal carcinoma, J. Nucl. Med. Allied Sci. 34 (1990) 9± 12. [25] A. Kostulak, The effect of ACTH and cortisone on the behaviour of arylsulphatase in the salivary glands of white rat, Acta Histochem. 58 (1977) 63±67. [26] L. Vitaioli, E. Baldoni, A. Cecchi, S.R. Indraccolo, Menstrual cycle-associated changes of sulphatides and arylsulphatase A activity in human uterine endometrium, J. Gynaecol. Obstet. 1 (1992) 1±4.
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[27] L. Vitaioli, A. Gobbietti, E. Baldoni, Arylsulphatase A activity and sulphatide concentration in the female rabbit oviduct are under physiological hormonal in¯uence, Histochem. J. 28 (1996) 149±156. È ner, S. Bekpõnar, F. C [28] P. O Ë õnar, A. Argun, Relationship of some endogenous sex steroid hormones to leukocyte arylsulphatase A activities in pre- and postmenopausal healthy women, Horm. Metab. Res. 26 (1994) 265±312. [29] L. Vitaioli, E. Baldoni, L. Bellini, Arylsulphatase activity in the oviduct of the frog Rana esculenta. I. Oestradiol-induced changes following ovariectomy and hypophysectomy, Histochem. J. 19 (1987) 217±224.
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Evaluation of leukocyte arylsulfatase-A activity in patients with breast cancer and benign breast disease È ner b,*, FuÈsun CËõnar b, Hikmet KocËak b, GuÈvencË GuÈvenen c, Sembol TuÈrkmen a, Pernur O HuÈseyin Altun d, Yavuz Eryavuz e a Department of Biochemistry, Social Insurance Institution, Okmeydanõ Educational Hospital, Istanbul, Turkey Department of Biochemistry, Istanbul Faculty of Medicine, University of Istanbul, CËapa 34390, Istanbul, Turkey c Department of Biochemistry, Social Insurance Institution, Istanbul Educational Hospital, Istanbul, Turkey d Department of General Surgery, Social Insurance Institution, Istanbul Educational Hospital, Istanbul, Turkey e Department of General Surgery, Social Insurance Institution, Okmeydanõ Educational Hospital, Istanbul, Turkey b
Received 15 November 2000; received in revised form 25 January 2001; accepted 29 January 2001
Abstract This study was planned to evaluate the feasibility of using the assay of leukocyte arylsulfatase-A (AS-A) activity as a noninvasive diagnostic tool in patients with benign and malignant breast disease. The leukocyte AS-A activity of a total of 81 women was analyzed, including 28 healthy women, 29 women with benign breast disease (BBD) and 24 patients with primary breast cancer (BC). The mean leukocyte AS-A activity in patients with BBD was slightly higher (14.3%) that observed in the healthy subjects, but the difference was not statistically signi®cant. In patients with BC the enzyme activity was signi®cantly higher than in the healthy subjects (60.3%, P , 0:05) and in the benign group (40.2%, P , 0:05). In addition, since no signi®cant differences have been observed between premenopausal patients and their controls, it is suggested that the measurement of leukocyte AS-A activity may not be a reliable test for differential diagnosis of benign and malignant proliferation in mammary glands due to the possible interfering effect of gonadal hormones on AS-A activity. In contrast, since peri- and postmenopausal BC patients have negligible or no gonadal activity function, the elevation in the activity of leukocyte AS-A in these age groups of patients may only be expected to originate from malignant proliferation. Based on our results, it is concluded that in patients in whom high leukocyte AS-A activities were observed the possibility of the presence of malignancy might also be high. Therefore, this test might be valuable as a non-invasive biochemical technique in combination with other established markers for the identi®cation of masses in the breast. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Leukocyte arylsulfatase-A; Benign breast disease; Breast cancer
1. Introduction Metabolic and especially anabolic processes are intensi®ed in the neoplasm and this manifests itself as a changed activity of a number of enzymes. More * Corresponding author. Tel.: 190-212-6311323-120; fax: 190212-6311323-115. È ner). E-mail address: [email protected] (P. O
than four decades ago, it was ®rst suggested that increased activity of arylsulfatases (ASs E.C.3.1.6.1.), which catalyze the hydrolysis of aromatic sulfate esters in malignant tissues [1] and also in the urine of patients with various active cancer [2,3], might be of diagnostic value. Since then, biochemical enzyme analyses have attracted great attention for better understanding of the biological
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00429-3
96
S. TuÈrkmen et al. / Cancer Letters 166 (2001) 95±101
nature of malignancies. Results of some previous studies indicated that measurement of arylsulfataseA (AS-A), a primary lysosomal enzyme, might be useful in the diagnosis and/or monitoring of the malignancy [4±10]. On the other hand, breast cancer (BC) is now recognized as probably the most prevalent malignant transformation occurring in women. In order to investigate the diagnosis and characterization of masses in the body with the aid of non-invasive biochemical techniques, BC is used as a best representative model [11]. There are limited reports concerning the activity of leukocyte AS-A in patients with several malignant tumors, but no such data in patients with BC are available to date. In the light of these data, this study was planned to investigate to what extent leukocyte AS-A activity could serve as an appropriate and reliable non-invasive diagnostic tool for malignant and benign cell proliferation in patients with newly diagnosed BC and benign breast disease (BBD), and also to provide some contribution to very limited data on this subject. 2. Materials and methods A total of 81 women participated in this study. The patient population consisted of 53 women who were admitted to the Department of General Surgery of the Istanbul Educational Hospital of the Social Insurance Institution because of pain or a palpable nodule in their mammary tissue. Based on their histopathology and/or ultrasonographic or mammographic examination, 29 of 53 patients ranging in age from 20 to 59 years were grouped as BBD and the remaining patients with ages ranging from 31 to 67 years were grouped as BC. Twenty-three of the BBD patients had ®brocystic disease, three had ®broadenoma, one had adenosis, one had lipomatosis and one had both lobular granulomatous mastitis and ®brocystic disease. Of the 24 newly diagnosed, untreated BC patients, 17 had only invasive ductal carcinoma, two had invasive ductal carcinoma with tubular differentiation, three had only lobular carcinoma, one had invasive ductal carcinoma with osteoclastic giant cells and one had only mucinous carcinoma. As a control group, 28 healthy women with ages ranging from 25 to 65 years and with no evidence of any type of cancer or
in¯ammatory disease were studied. None of the study population had hormonal or drug therapy including vitamin supplementation. Subjects with a history of an allergic or anaphylactic reaction and pregnant or lactating women were excluded from the study. The participants were not restricted to a speci®c diet. Informed consent was obtained from each participant. Menopausal status at diagnosis was not speci®ed by hormone analysis, but women with ages ranging from 20 to 37 years who had regular menstrual cycles were labeled as premenopausal. Postmenopausal women were de®ned as those who had no menstrual bleeding in the 3 years prior to study and women with ages ranging from 38 to 50 years were designated as perimenopausal. Blood samples from patients with BC or BBD were obtained prior to any surgical or other therapeutic procedures. Fasting venous blood (11 ml; 10 ml of it anticoagulated with Heparin (Liquemine, Roche) and the remainder with EDTA) was drawn from each subject. Leukocytes were prepared from whole blood by adding an equal volume of Dextran (Sigma) in 0.85% saline and AS-A activity in leukocytes was measured using p-nitrocathecolsulphate (Sigma) as a substrate by the method of Pilz [12]. The amount of liberated nitrocathecol (NC) was determined by absorption at 515 nm in a LKB ultraspec 4050 spectrophotometer. Enzyme activity was expressed as nanomoles of hydrolyzed p-nitrocathecol per milligram of protein per hour at 378C. Protein was determined by the Lowry procedure [13] using serum albumin as the standard. All assays were run in duplicate and the results were the mean of duplicate measurements. The results for BC patients were compared with the results for healthy subjects and patients with BBD. Venous blood (1 ml) anticoagulated with EDTA was used for white blood cell counts using a Coulter JS counter. Comparisons between the healthy subjects and patients were performed using one-way ANOVA followed by a post-hoc Tukey HSD test and probability values of 0.05 or less were considered signi®cant. Differences in leukocyte AS-A activity between the age groups were tested using the Kruskal±Wallis test. The software used for all statistical evaluations was the SPSS 7.5 statistical package program. To estimate the validity of AS-A, sensitivity, speci®city and predictive values of the test were calculated.
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Table 1 Leukocyte AS-A activity in patients with benign and malignant breast disease and in healthy women (mean ^ SD)
Leukocyte AS-A (nmol NC/mg protein per h) a
Healthy women (n 28)
BBD patients (n 29)
BC patients (n 24)
83.6 ^ 16.8
95.6 ^ 27.6
134.0 ^ 52.0 a
P , 0:05 compared to control and BBD groups (Tukey HSD multiple comparison test).
The cut-off level for AS-A was established as 127.0 nmol NC based on leukocyte levels excluding approximately 97% of the healthy controls (this level is approximately equivalent to the mean 1 2.5 SD derived from the data). 3. Results Leukocyte AS-A, activities for normal subjects, patients with histologically con®rmed BC and patients with BBD are shown in Table 1 and Fig. 1. Leukocyte AS-A activities in healthy women, and in patients with BBD and BC ranged from 64.0 to 127.0 (mean 83.6 ^ 16.8), from 51.0 to 148.0 (mean 95.6 ^ 27.6), and from 68.0 to 256 (mean 134.0 ^ 52.0) nmol NC/ mg protein per h, respectively. Although the lower and upper enzyme activity levels in BBD and BC
Fig. 1. Distribution of leukocyte AS-A activity in normal women and patients with BBD and BC and values of the main statistical parameters.
patients were more widely distributed, the mean values observed in both non-malignant and malignant groups were found to be approximately 15 and 60% higher, respectively, than those of the control group. The difference reached statistical signi®cance in BC patients (P , 0:05, Table 1). In addition, leukocyte AS-A activity in patients with BC was 40.2% higher that observed in BBD patients, being the difference at the level of P , 0:05 (Table 1). The groups were divided into three subgroups according to their gonadal activity status as: premenopausal, aged 20±37 years (n 20); perimenopausal, aged 38±50 years (n 30); and postmenopausal, aged 51±67 years (n 31). The mean leukocyte ASA activities in patients aged 20±37 years with benign and malignant breast disease were not signi®cantly different from those observed in aged-matched controls (Table 2). In patients with BBD and BC aged between 38 and 50 years, the mean levels of leukocyte AS-A activity were found to be elevated compared with agedmatched controls, but only the difference between the levels of AS-A activity for BC patients and the control group was statistically signi®cant at a borderline level (P 0:056). The difference between the groups did not exhibit a high degree of signi®cance due to the small number of cases in both groups. The highest leukocyte AS-A activity was found in BC patients aged 51±67 years as compared to BC patients in pre- and perimenopausal categories. The mean leukocyte AS-A activity in postmenopausal BC patients aged 51±67 years was signi®cantly higher than those of aged-matched controls and BBD patients (P 0:001) (Table 2). The enzyme activity observed in postmenopausal BBD patients was found to be slightly but signi®cantly lower (P 0:001) than the aged-matched control group. When we used the upper limit of the normal range as 127 nmol NC/mg protein per h for leukocyte AS-A,
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Table 2 Leukocyte AS-A activities (nmol NC/mg protein per h) in patients and in healthy women at different ages (mean ^ SD) a Age ranges (years)
Healthy women
BBD patients
BC patients
20±37 38±50 51±67
111.0 ^ 15.8 (5) 79.5 ^ 12.0 (7) 75.6 ^ 9.8 (16)
99.8 ^ 22.1 (11) 98.9 ^ 30.4 (15) 63.6 ^ 6.6 (3)*
94.2 ^ 20.9 (4) 127.8 ^ 42.4 (8) 151.5 ^ 58.8 (12)*
a
The number of subjects is in parentheses. *P 0:001 compared to control and BBD groups.
inconclusive because of the lower level of the enzyme in blood than in urine [6,14,15]. Nevertheless, human neutrophil leukocytes are rich sources of ASs and peripheral circulating leukocytes often provide a convenient nucleated cell model for studying the biochemistry and pathology of other nucleated cells in the body [16,17]. Therefore, we preferably investigated the AS-A activity in peripheral leukocytes obtained from newly diagnosed, untreated BC patients and from BBD patients. The upper and lower levels of leukocyte AS-A activity in the method used by us are reported as 126.0 and 52.4 nmol NC/mg protein per h, respectively, with a mean value of 72.3 nmol NC [12]. Thus, our values observed for healthy subjects and for the majority of BBD patients (86.3%) are well within the reported values of Pilz [12] and those of other investigators [9,12,18±20]. The mean leukocyte AS-A activity value in patients with BBD did not differ signi®cantly from controls. This ®nding is similar to that obtained in a previous study reporting that AS-A activity in ®brocystic tissue did not differ distinctly from that found in normal tissue [4]. Our ®nding of a non-signi®cant difference between the mean value of leukocyte AS-A for normal subjects and for BBD patients suggested that the measurement of leukocyte AS-A may not be valuable for the diag-
the activity of 1/28 (3.5%) healthy women, 4/29 (13.7%) patients with BBD and 12/24 (50%) patients with BC was at or above this cut-off level. The sensitivity, speci®city and predictive values of a positive result were 0.50, 0.96, and 0.92, respectively, at the 127 nmol NC cut-off level. The overall test accuracy was 48.1% (Table 3). 4. Discussion In recent years, there has been expanding interest in the use of normal antigens, hormones, and enzymes for the diagnosis and prognosis of tumors and also for the assessment of treatment response. However, very few published clinical studies of the value of leukocyte AS-A activity in BC patients are available to date. In one of the preliminary studies performed on a very small number of BC patients, the activity of ASA was found to be signi®cantly increased in mammary tissues of three of ®ve BC patients examined, but in two cases pretreated with X-ray and in two BBD patients the level of the enzyme was found to be normal [4]. Analysis of the blood level of AS-A in several pathological states is scanty and results are generally
Table 3 Sensitivity, speci®city and predictive values of the leukocyte AS-A activity a Sensitivity
A B a
0.50 0.62
Speci®city
0.96 0.82
Predictive values
Overall test accuracy
(1)
(2)
0.92 0.75
0.69 0.60
0.48 0.46
Values are shown at 127 nmol NC (A) and 100.4 nmol (B) cut-off levels. The cut-off level 100.4 nmol NC is derived from data based on the mean 1 1 SD.
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nosis of BBD. In contrast, the more pronounced activity of leukocyte AS-A in BC patients than in healthy subjects (60.3%) and BBD patients (40.2%) strengthened the conclusion that the measurement of enzyme activity may be useful in the differential diagnosis of benign and malignant proliferation. In addition, our ®ndings concerning the value of variation coef®cients for AS-A activity in controls and patients with BC is similar to the ®ndings of Laidler et al. [7], who also found a 25.5% variation coef®cient for the serum ASA concentration in patients with BC. To the best of our knowledge, this is the ®rst report concerning the measurement of leukocyte AS-A activity both in patients with BC and with BBD. Consequently, there are no available data for direct comparison with our results. However, highly increased leukocyte AS-A activities in leukemia patients have been previously reported [9,21]. High leukocyte AS-A activity in BC patients observed by us suggested that patients having high AS-A activity in leukocytes and/or tumor tissues might be associated with malignant disease. The ®ndings of previous studies reporting elevated levels of AS-A in solid tumor tissues, leukocytes and the urine of patients with various cancers seem to support our suggestion [4,5,8,9,21± 23]. The activity of leukocyte AS-A in patients with BC was increased above the upper limit of the normal range (127 nmol NC) in 50% of cases. This result is comparable with the result obtained in an earlier work reporting that an elevated value for CA 15.3 over the upper cut-off level was also observed for 48.7% of BC patients before surgery [24]. However, on the basis of this ®nding it is concluded that the presence of the tumor should not be excluded when the tumor marker levels are below the cut-off point. Accordingly, because half of our BC patients had a value of leukocyte AS-A activity above the upper limit of the normal range, it should not be thought that measurement of leukocyte AS-A activity is not suf®ciently valuable for suspecting the presence of malignancy. In addition, 66% of the remaining BC patients were above the mean level of leukocyte AS-A activity for healthy women. Previous research on lysosomal enzymes including ASs and their possible connection with endocrine functions strongly suggest the in¯uence of hormonal factors on AS-A activity [23,25±29]. In one of these
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studies, increased leukocyte AS-A activity in healthy premenopausal women during ovulatory and luteal phases was observed [28]. In the other studies, elevations in both AS-A and B activities in experimental animals during physiological estrus was observed [27,29]. The results of these observations suggest that elevations in leukocyte and urine AS-A activities may be observed without any pathological cause under physiological or experimental conditions, especially in hyperestrogenic subjects [28]. Furthermore, in a previous study performed on human endometrium in which proliferation and differentiation are controlled by sex hormones, the levels of AS-A in endometrium biopsy samples from women with cystic hyperplasia or adenocarcinoma were also found to be higher, being more pronounced in the neoplastic endometrium than those obtained from normal women in the proliferative phase [23]. On examination of the three subgroups which were divided according to their gonadal activity status as pre-, peri-, and postmenopausal groups, the mean values of leukocyte AS-A activities in premenopausal benign and malignant breast disease patients were not found to be signi®cantly different than those of agematched controls. This ®nding may be due to the randomness of the time of blood collection and perhaps it is at the time of the menstrual period that gonadal steroids are in¯uential. The reason for not standardizing the blood collection time was the obligation to obtain blood specimens from carcinoma patients prior to surgical intervention. Therefore, one might suggest that in women having in¯uential gonadal activity function, the measurement of leukocyte AS-A activity may not provide a reliable test for use in the differential diagnosis of benign and malignant proliferation in mammary glands. Since perimenopausal BC patients have little gonadal activity function and postmenopausal patients have absolutely no gonadal function, the elevations in the activity of leukocyte AS-A in BC patients between 38±50 and 51±67 years of age in our study may only be expected to originate from neoplastic proliferation. In addition, it should be of considerable interest to investigate ASA activity in BC patients treated with the antiestrogenic drug Tamoxifen. The lack of statistical signi®cance between perimenopausal BC and BBD patients aged between 38 and 50 years may be due to the smaller sample size and reduced statistical power.
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In our study, the highly signi®cant AS-A concentration in leukocytes of BC patients could have resulted from intensi®ed cell metabolism and/or stimulated synthesis of the enzyme due to a cytokine produced by malignant cells or their surrounding cells. In discriminating healthy cases from patients with BC using the AS-A cut-off limit value, only 1/28 healthy individuals were false positive and 12/24 BC patients were false negative, leading to a speci®city of 0.96 and a sensitivity of 0.50. Accordingly, the test seems to be able to detect 50% of BCs with a speci®city approaching 100%. In addition, our ®ndings of an overall spectrophotometric test accuracy of 48.1% and positive and negative predictive values of 92.3 and 69.2%, respectively, suggested that measurement of leukocyte AS-A might provide a helpful noninvasive test for the diagnostic work-up. From the results obtained in our study, it can be concluded that in patients having high leukocyte AS-A activity, the probability of the presence of malignancy might also be high, and therefore this test might be valuable as a non-invasive biochemical technique in combination with other established markers for the identi®cation of masses in the breast. Acknowledgements The authors would like to thank Drs Nurten Turan È mit TuÈrkogÏlu for their help in data analyses. and U References [1] E. Boyland, D.M. Wallace, D.C. Williams, The activity of the enzymes sulphatase and b-glucuronidase in the urine, serum and bladder, Br. J. Cancer 9 (1995) 62±67. [2] L.M. Dzialoszynski, The clinical value of arylsulfatase estimation in urine, Clin. Chim. Acta 2 (1957) 542±547. [3] L.E. Posey, L.R. Morgan, Measurement of arylsulphatase A activity in urine, Clin. Chem. 25 (1979) 328±331. [4] L.M. Dzialoszynski, J.L. Kroll, A. FroÈchlich, Arylsulphatase activity of some malignant tumors, Clin. Chim. Acta 14 (1966) 450±453. [5] S. Gasa, A. Makita, T. Kameya, T. Kodama, E. Araki, T. Yoneyama, M. Hirama, M. Hashimoto, Elevated activities and properties of arylsulfatase A and B variant in human lung tumors, Cancer Res. 40 (1980) 3804±3809. [6] K. Honke, T. Fujii, A. Makita, Arylsulfatase A protein in sera of cancer patients, Cancer J. 8 (1995) 139±142.
[7] P. Laidler, D. Kowalski, J. Silberring, Arylsulphatase A in serum from patients with cancer of various organs, Clin. Chim. Acta 204 (1991) 69±78. [8] K. Mitsuhashi, A. Maru, T. Kayanagi, T. Ishibashi, Y. Imai, S. Gasa, N. Taniguchi, A. Makita, Arylsulphatase A activities in urine and tissues taken from bladder cancer patients, Jpn. J. Exp. Med. 54 (1984) 211±216. È mer, P. O È ner, M. Manyaslõ, M. Aktan, Leukocyte aryl[9] B. O sulphatase A activity in acute leukemia, Cancer Biochem. Biophys. 14 (1994) 53±56. [10] L.E. Posey, J. Rainey, R. Vial, D. Ryan, J.H. Bickers, R.L. Morgan Jr., M.S. Samuels, E.W. Hull, A noninvasive technique for monitoring response to chemotherapy in human acute leukemia, Cancer 44 (1979) 873±880. [11] G.F. Schwartz, R. Schwarting, P. Rabindranauth, G.C. Finkel, Clinical applications of serum and tissue markers in malignant disease: breast cancer as a paradigm, Clin. Chem. 39 (11) (1993) 2404±2412. [12] H. Pilz, Late adult metachromatic leukodystrophy, Arch. Neurol. 27 (1972) 87±90. [13] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265±275. [14] A.J. Gniot-Szulzycka, Arylsulphatase A and B in blood, Clin. Chim. Acta 32 (1971) 17±23. [15] P.M. Laidler, K.W. Ryder, M.R. Glick, T.O. Oei, R.L. Van Etten, Radioimmunoassay for arylsulphatase A in urine, Clin. Chem. 31 (1985) 391±396. [16] M.C. Geokas, H. Rinderknecht, Plasma arylsulphatase and bglucoronidase in acute alcoholism, Clin. Chim. Acta 46 (1973) 27±32. [17] P. Martinelli, M. Montanari, M. Ippoliti, M. Mochi, S. Sangiorgi, M. Capocasa, Familial spasmodic dysphonia with low arylsulphatase A (AS-A) level, Acta Neurol. Scand. 91 (1995) 196±199. [18] B. Herz, G. Bach, Arylsulphatase A in pseudode®ciency, Hum. Genet. 66 (1984) 147±150. [19] M.J.A.J.M. Hoes, K.J.B. Lamers, R.A. Wevers, Use of arylsulphatase A in chronic psychiatric patients, Am. J. Psychiat. 145 (1988) 382. [20] P. Propping, W. Friedl, M. Huschka, K.H. SchloÈr, F. Reimer, M. Lee-Vaupel, E. Conzelmann, K. Sandhoff, The in¯uence of low arylsulphatase activity on neuropsychiatric morbidity: a large-scale screening in patients, Hum. Genet. 74 (1986) 244±248. [21] Y. Uehara, S. Gasa, A. Makita, K. Sakurada, T. Kiyazaki, Lysosomal arylsulfatase of human leukocytes. Increment of phosphorylated B variants in chronic myelogenous leukemia, Cancer Res. 43 (1983) 5618±5622. [22] N. Ueno, T. Matsumoto, M. Ohkawa, M. Nakayama, T. Tonoka, Urinary arylsulphatase in normal children and in patients with pediatric malignant disease, Tohoku J. Exp. Med. 139 (1983) 165±169. [23] L. Vitaioli, E. Baldoni, R. Ricci, S.R. Indraccolo, Expression of acidic glycosphingolipids and arylsulphatase A activity in human pathological endometrium, Eur. J. Obstet. Gynecol. Reprod. Biol. 54 (1994) 31±35.
S. TuÈrkmen et al. / Cancer Letters 166 (2001) 95±101 [24] M. Gion, G. Ruggeri, R. Marconato, C. Casella, A. Nosadini, G. Laffranchini, R. Mione, S. Belloli, G. Bruscagnin, Perioperatory kinetic evaluation of tumor markers in breast and colorectal carcinoma, J. Nucl. Med. Allied Sci. 34 (1990) 9± 12. [25] A. Kostulak, The effect of ACTH and cortisone on the behaviour of arylsulphatase in the salivary glands of white rat, Acta Histochem. 58 (1977) 63±67. [26] L. Vitaioli, E. Baldoni, A. Cecchi, S.R. Indraccolo, Menstrual cycle-associated changes of sulphatides and arylsulphatase A activity in human uterine endometrium, J. Gynaecol. Obstet. 1 (1992) 1±4.
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[27] L. Vitaioli, A. Gobbietti, E. Baldoni, Arylsulphatase A activity and sulphatide concentration in the female rabbit oviduct are under physiological hormonal in¯uence, Histochem. J. 28 (1996) 149±156. È ner, S. Bekpõnar, F. C [28] P. O Ë õnar, A. Argun, Relationship of some endogenous sex steroid hormones to leukocyte arylsulphatase A activities in pre- and postmenopausal healthy women, Horm. Metab. Res. 26 (1994) 265±312. [29] L. Vitaioli, E. Baldoni, L. Bellini, Arylsulphatase activity in the oviduct of the frog Rana esculenta. I. Oestradiol-induced changes following ovariectomy and hypophysectomy, Histochem. J. 19 (1987) 217±224.