Lee Roy Morgan, Jr., Departments of. Pharmacology and Medicine ... Morgan et al. (13) measured ar-. Presented ... and Haas, Philadelphia,. PA 19105) per liter.
CLIN. CHEM. 25/2, 328-331
(1979)
ipp©cxll ll#{174}1 J1b©IL
Measurement of ArylsulfataseA Activity in Urine1 Submitters:
Evaluators:
Lynn E. Posey, Department of Pathology, Louisiana State University Medical Center, New Orleans, LA 70112 Lee Roy Morgan, Jr., Departments of Pharmacology and Medicine, Louisiana State University Medical Center, New Orleans, LA 70112 Sam Levin, Inst itut Jules Bordet, Centre des Tumeurs de L’Universit#{233}Libre de Bruxelles, Bruxelles, Belgium Morton K. Schwartz, Memorial-Sloan Kettering Cancer Center, New York, NY 10021
Introduction Arylsulfatase (aryl-sulphatase suiphohydrolase, EC 3.1.6.1) is any enzyme that hydrolyzes aromatic sulfates to the corresponding alcohol and sulfate(1). In humans, arylsulfatases have been found in every type of cell examined except the mature erythrocyte (2). Although their physiological functions are not immediately apparent, their ubiquity suggests an important biological role.
Three such enzymes-”arylsulfatases A, B, and C”-have been characterized and extensively studied. Although their names imply that they are isoenzymes, they actually are three distinct enzymes, differing from each other in pH optima, substrate affinities, reaction to ionic inhibitors, and subcellular localization. Decreased activity of arylsulfatase A has been well documented to be the primary defect in patients with metachromatic leukodystrophy. Austin et al. (3, 4) first noticed that patients with this disorder consistently had decreased arylsulfatase A activities in their tissues and urine. Later, Mehl and Jatzkewitz (5, 6) showed that cerebroside-3-sulfate was a physiological substrate for arylsulfatase A. Other sulfate glycolipids have also been shown to be substrates for this enzyme (7, 8). Arylsulfatase activities in biological materials increase in physiological and pathological conditions such as pregnancy, wound healing, inflammatory processes, intoxications, and malignant disease (9). Dzialoszynski (9, 10) believed that increased arylsulfatase activity in the urine was an indication of the presence of a malignant disease or an inflammatory process. Urinary arylsulfatase activities are increased in patients with transitional cell carcinoma of the bladder (11) and have been implicated in the pathogenesis of bladder cancer for the role they may play in releasing suspected carcinogens in the urinary bladder from their detoxified sulfate conjugates (12). Morgan et al. (13) measured arPresented in part at the Fall Meeting of the Southeast of the AACC, Birmingham, AL, October 1975.
328
CLINICAL CHEMISTRY, Vol. 25, No. 2. 1979
Section
ylsulfatase activities in urine from patients with colorectal carcinoma and found the results to be useful as a prognostic indicator: arylsulfatase activity either increased as the disease progressed or decreased during clinical responses to the administration of antineoplastic drugs. Similar observations have been reported for patients with acute leukemia (14). To date, the highest urine arylsulfatase activities have been found in patients with acute leukemia, 30-fold increases over normal being observed (9, 10). An automated assay for arylsulfatase A activity has been developed because of its apparent clinical usefulness in the diagnosis of metachromatic leukodystrophy and as a prognostic indicator in malignant disease. The automated assay is based on the widely used method of Baum et al. (15). Methods commonly used to determine arylsulfatase activity involve the quantitation of the desulfated phenolic residue released when an arylsulfate is used as the substrate (16). Dodgson and Spencer (17) reviewed the insensitive and tedious methods for quantitating the sulfate ions that are liberated in the reactions catalyzed by the arylsulfatases.
Principle Figure lisa schematic diagram of the principle underlying the determination of arylsulfatase activity with potassium 4-nitrocatechol sulfate (potassium 2-hydroxy-5-nitrophenyl sulfate) as the substrate. Arylsulfatases A and B can be determined in the presence of each other by exploiting their different inhibition by inorganic ions, their different pH optima, and their different susceptibility to substrate concentration. The pH optima for arylsulfatases A and B are 5.0 and 6.0,respectively (15). In the determination of arylsulfatase A, sodium chloride is used to inhibit the activity of arylsulfatase B (15).
Materials and Methods Reagents 1. Acetate buffer, 0.5 moh/L, pH 5.0. This reagent contains, per liter, 5 X 10 mol of sodium pyrophosphate .10 H90 and 1.71 mol of sodium chloride. 2. Dipotassium 4-nitrocatechol sulfate solution, 0.01 mollL. In a 100-mL volumetric flask, dissolve 311.4 mg of potassium 4-nitrocatechol sulfate (Sigma Chemical Co., St. Louis, MO 63178) in 0.5 mol/L acetate buffer, pH 5.0, containing, per liter, 5 X 10 mol of sodium pyrophosphate and 1.71 mol of sodium chloride. This solution is stable for four weeks in a brown-glass container at -20 #{176}C. 3. Sodium hydroxide, approximately I mollL. 4. Wash water, for the automated method, containing 1 mL of surfactant
(Triton
PA 19105) per liter.
X-100;
Rohm
and Haas,
Philadelphia,
oil
-
HO
pH5O HO ArylsutfataseA N’O ROil SO
__.
‘iIi.._.
0
Double
0
_!I2!...
coil hsatmg
Oy
bath
NO 0
0 4-Nilrocafechol
___________
2-hydrouy-5nitrophenyl sulfate (4-Nitrocafechol sulfate)
-NtTRocArEcIioL
YELLOW
YELLOW
RED
Fig. 1.Schematic presentationofthe reactioncatalyzedby ar-
ylsulfatase A Specimen
Collection
and Storage
Collect a 24-h urine2 with no preservative. The urine should be refrigerated during collection. No special diet is required before the specimen is collected. The enzyme activity is stable for three days at 4 #{176}C; centrifuged aliquots can be stored for no longer than three weeks at -20 #{176}C. Note: Evaluator S.L. found that arylsulfatase A activity in samples of dialyzed urine was stable for as long as 18 days at -20 #{176}C.
15mmtubular flow cell 520nm
Fig. 2. Flow diagram for the AutoAnalyzer
Note: Evaluator
S.L. measured the absorbances in a Zeiss PMQ II spectrophotometer, at 510 nm. Readings with this instrument can be converted to absorbances that would be obtained with a Beckman DU spectrophotometer by multiplying the reading by 1.09.
Procedure3
Calculations
1. Centrifuge a 10-mL aliquot of urine. 2. Carefully pipet 4.0 mL of urine into a dialysis bag (Fisher Scientific, 30 cm X 1 cm) that has been presoaked in de-ionized water.
Enzymatic activity is expressed in terms produced by the following expression:
Note:
Evaluator
S.L. used Sigma dialysis
sacks (cat. no.
N250-7), cut in half. 3. Dialyze, with stirring, against de-ionizedwater for at least 18 h at 4 #{176}C. Use 500 mL of de-ionized water for each sample. 4. Empty the urine from the dialysisbag into a 6-mL graduated test tube. Rinse the bag with 1 mL of de-ionized water and add the rinse to the dialyzed urine. Dilute the sample to 6 mL with de-ionized water.
Note: Evaluator
S.L. emptied the urine into a 12- or 15-mL graduated tube and diluted to 9 mL rather than 6 mL, to decrease the absorbance after the reaction was completed. Even so, further dilution was sometimes necessary.
5. Add 1 mL of prewarmed (37 #{176}C), dialyzed urine to 1 mL of prewarmed (37 #{176}C) substrate solution. After exactly 60 mm at 37 #{176}C, stop the reaction and develop the color by adding 0.5 mL of 1 mol/L sodium hydroxide. Prepare a blank with the same volumes of sample and substrate, but incubate separately for the same time period and mix after adding the sodium hydroxide to the substrate solution. 6. Measure the absorbances at 510 nm within 1 h. We used the Coleman Jr. II Model 6/20 spectrophotometer. Readings with the Coleman can be converted to absorbances that would be obtained with a Beckman Model DU spectrophotometer by multiplying the reading by 1.18. 2
Twenty-four-hour urines are collected because there is a diurnal in enzyme activity (13). The method of Baum et al. (15) was modified to increase sensi-
variation
tivity. The volume of sodium hydroxide used to stop the reaction and develop the color was decreased so that the final volume of the reaction mixture was half that obtained by the original procedure, thus doubling the absorbance. This conversion factor was obtained from a linear regression analysis on data obtained by reading the absorhance of 50 samples withboth instruments. The correlation coefficient was 0.99.
(A
-
of the product
A,,) X 5.53
where A is the absorbance of the test solution and A,, the absorbance of the blank solution. The factor 5.53 is derived as follows: Activity, U/L
=
[(As
-
A,,) x 2.5 X 1.5 X 1031/(11.3X 60
x
1)
where 2.5 is the dilution factor for the reaction mixture, 1.5 is the dialysis dilution factor, 10 converts U/mL to UTL, 11.3 is the apparent molar absorptivity of 4-nitrocatechol (mL umol cm-’), 60 is the duration in minutes, and 1 is the length of the light path in centimeters. Express the activity in terms of the 24-h volume.
Note: Evaluator
S.L.’s dialysis dilution factor is 2.25. Enzymatic activity is then obtained by the following expression: (A - A,,) X 8.3.
Continuous-Flow
Mechanized
Procedure
Figure 2 shows the flow diagram for the AutoAnalyzer. Sampling rateis40 specimens per hour, with a sample/wash ratio of 1/2. Standards and samples are mixed with substrate in a standard 14-turn mixing coil before entering a double-coil heating bath at 37 #{176}C. The reaction is stopped and the color developed by adding sodium hydroxide, which is mixed in a 28-turn standard mixing coil before entering the debubbler. The colored solution is measured in a 15-mm tubular flow cell at 520 nm. The recorder traces the color intensity as a peak on chart paper printed in percent transmittance. The AutoAnalyzer system was standardized with solutions of pooled dialyzed normal urine of known enzymatic activity, as determined by triplicate manual assays. 1. Steps 1 through 4 are identical to the manual procedure described above. 2. With the reagent and sample lines in wash water, set the baseline of the recorder to zero absorbance. 3. Occlude the light path and set the recorder to zero percent transmittance. 4. Express the enzyme activity (in SI units) by referring to CLINICAL CHEMISTRY, Vol. 25, No. 2, 1979
329
3.5
12 I#{176}’”w1 range
10
I.e
S
0.8
$
F
S
S
Fig. 3. TypIcal tracing from the AutoAnalyzer a standard curve. A typical tracing and the resulting
standard in Figures 3 and 4, respectively. 5. Express the activityin terms of the 24-h volume.
curve
are
“.4
given
2
A pooled specimen of urine, centrifuged and stored at -20 #{176}C for no longer than three weeks, should be analyzed for quality control. Arylsulfatase activity is reported to be stable at 4 #{176}C in 3.2 mol/L ammonium sulfate for several months
S
S
. #{149}
8
.
S
I
1.
Ab’mo/i#3
I
I
Colon
I
I
LetA
Breast
I
Blad.
I
Lung
Other
Type of molignancy Fig. 5. Urinary arylsulfatase A activity malignancies The shaded area indicates the normal range
in patients with various
mission from prior chemotherapy or surgery had urinary arylsulfatase A activities that were within the normal range. Note: Evaluator S.L. obtained urine specimens from 20 patients with neoplastic disease of various tissues and organs (breast, lung, kidney, leukemias, and lymphomas). Of the 20, 13 (65%) had increased activities. Of five patients on a neurosurgical ward, one patient with major
Note: Evaluator S.L. examined 14 normal individuals, male and female, ages 18 to 55, and found the normal range to be 0.7-4.3 U/day (mean, 2.5 U/day; SD, 0.9).
cranial
contusions
activity
Values in Disease Urine specimens from patients with various types of cancers were assayed as described for arylsulfatase A activities (Figure 5). Of 80 patients with active disease, 63 (79%) had increased urine arylsulfatase A activities. When the patients were grouped according to the extent of disease, 61% of the patients with localized disease (primary tumor with or without regional lymph node involvement) showed increased activity of urine arylsulfatase A; 92% of patients with distant metastases had above-normal activities. Four of five patients in clinical re-
S
8
8
and we have added it to solutions of pooled dialyzed human urine that are to be used for quality control. This preparation isstableforat leasttwo months.
Normal ranges were established by measuring the activities of the enzymes in urinesfrom 30 apparently normal individuals who were free from any acute or chronic disease process. The study included 16 women (16-73 years old) and 14 men (22-38 years old). Normal ranges, calculated as the mean ±2 SD, for the manual and the mechanized methods were 0.3-1.4 U/day and 0.3-1.2 U/day, respectively.
.
.5
(18),
Normal Ranges
S
S
3
Quality Control
$
showed
increased
urine
arylsulfatase
A
(13.4 U/day).
Discussion Arylsulfatase A activity can be determined simply and reproducibly. Table 1 summarizes within-day and day-to-day precision for the manual and mechanized methods. Precision data and CVs are similar for the two methods. Absorbance increased linearly with enzyme activity to at least an absorbance of 0.90, which corresponds to an activity
Table 1. Precision of Determination of Arylsulfatase A in Urine, Low- and High-Value Samples Manual
method
Low
HIgh
Mechanized Low
method HIgh
Within-day: n Mean, U/L SD, U/L
10 0.49
10 3.41
10 0.31
10 3.01
0.05
CV, %
10.2
0.24 7.0
0.04 12.9
0.23 7.6
11 3.16 0.23 7.3
11 0.35 0.04 11.4
10 3.32 0.26 7.8
Day-to-day:
10
n
2 Arylsulfotase A activity
3
4 (mU/mi)
Fig. 4. Standard curve resulting from the tracing in Figure 3 330 CLINICALCHEMISTRY,
Vol. 25, No. 2, 1979
Mean, U/L
SD, U/L CV
%
0.43
0.05
11.6
of 5
U/L. Blanks were unnecessary for the automated method.
Readable
blank peaks were obtained
only infrequently
(3/53)
and did not significantly affect the results. Carryover was negligible. Results by the manual and the mechanized methods correlated highly (r = 0.98). The major drawback to the continuous-flow assay is that the dialysis is not mechanized. The usual mechanized dialysis system used in AutoAnalyzers is not sufficient to remove the interfering ions, even with two sets of dialysis plates used at 37 #{176}C. Arylsulfatase A is deficient in the tissues and body fluids of patients with metachromatic leukodystrophy (3-5,8). Increases have been seen in malignant tumor tissue (19), in sera from patients suffering from acute alcoholic intoxication (20), isopropanol and arsenic poisoning (21), and kwashiorkor (22), and in urine specimens collected from patients with malignancies (10, 13) and inflammatory processes of the genitourmary tract (10). Increases were only slight in Hodgkin’s disease, heart diseases (valvular lesions, endocarditis, myocardial infarct), liver diseases (infectious hepatitis, cirrhosis), pulmonary tuberculosis, stomach ulcer, and gout. Wound healing is also accompanied by increased arylsulfatase activity in the urine. Arylsulfatase activity was determined in the urine of patients both before and after such surgical procedures as repair of a hernia, resection of the stomach (benign disease), and removal of goiters or varicose veins (10). In all patients, urine arylsulfatase activity was normal before surgery, increased after surgery as healing progressed, then gradually returned to normal after about a week (70). Values 10- to 15-fold those seen in controls were observed in urines from patients with lymphocytic leukemia or carcinoma of the breast, cervix, prostate, kidney, bladder, and skin
determination of phenosulfatase (arylsulfatase). Biochem. J. 48, xxciii (1951). Abstract. 2. DeDuve, C., and Wattieaux, R., Functions of lysosomes. Ann. Rev. Physiol. 28,435-492 (1966). 3. Austin, J., McAfee, D., Armstrong, D., et al., Abnormal sulphatase in two human diseases (metachromatic leukodystrophy and gargoylism). Biochem. J. 93, 15C-17C (1964). Abstract. 4. Austin, J., Armstrong, D., and Shearer, L., Metachromatic form sclerosis. V. The nature and significance of low A controlled study of brain, liver, and kidney in four patients with metachromatic leukodystrophy (MLD). Arch. Neurol. 13, 593-614 (1965).
of diffuse sulfatase
5. MehI,
cerebral
activity.
E., and Jatzkewitz,
metachromatic Commun.
H., Evidence
leucodystrophy
(ML).
for the genetic
Biochem.
block
Biophys.
in
Res.
19,407-411 (1965).
6. MehI, E., and Jatzkewitz, logical substrate 619-627 (1968).
H., Cerebroside 3-sulfateas a physioof arylsulfatase A. Biochim. Biophys. Acta 151,
Fluharty, A. L., Stevens, R. L., Miller, R. T., and Kihara, H., Sulfoglycerogalactolipid from rat testis: A substrate for pure human arylsulfatase A. F3iochem. Biophys. Res. Commun. 61, 348-354 (1974). 7.
8. Eto, Y., Wiesmann, U., and Herschkowitz, N. N., Sulfogalactosylsphingosine sulfatase. Characteristics of the enzyme and its deficiency in metachromatic leukodystrophy in human cultured skin 11broblasts.J. Biol. Chem. 249,4955-4960 (1974). 9. Dzialoszynski,L. M., and Gniot-Szulzycka,J., The clinical aspects of arylsulphataseactivity.Clin. Chim. Acta 15,381-386 (1967).
10. Dzialoszynski,L. M., The clinical value of arylsulfatase estimation in urine.Clin. Chim. Acta 2,542-547 (1957). 11. Posey, L. E.,and Morgan, L. R. Urine enzyme activities in patients with transitional cell carcinoma of the bladder. Clin. Chim. Acta 74, 7-10 (1977). 12. Boyland, E., Manson, D., Sims, P., and Williams, D. C., The enzymic hydrolysis of derivatives of aminophenols.Biochem. J. 59,
xix-xx(1955).
(10).
Morgan et al. (13) reported on arylsulfatase in patients with cancer of the large bowel. Cancer-tissue arylsulfatase can be used to predict patient response to therapy with 5-fluorouracil. Only patients whose large-bowel mucosa had low or normal arylsulfatase activities responded to therapy. In addition, patients who responded to therapy also had decreased urine arylsulfatase activity accompanying such therapy. If the disease recurred, the activity of the enzyme again increased in the urine. Urine arylsulfatase activity was correlated with titers of carcinoembryonic antigen in the plasma and with the amount of disease presented by Dukes’ classification (13). Determination of arylsulfatase A is necessary to confirm the diagnosis of metachromatic leukodystrophy. It is also of value clinically as a biochemical assay that can serve as an indicator of a patient’s status once the presence of malignant disease has been established, as Dzialoszynski (10) suggested over 20 years ago. Undoubtedly, other applications will be found.
13. Morgan, L. R., Samuels, M. S., Thomas, W., et al., Arylsulfatase B in colorectal cancer. Cancer 36, 2337-2345 (1975). 14. Posey, L. E., Rainey, J., Vial, R., et al., A noninvasive technique for monitoring response to chemotherapy in human acute leukemia. Cancer, in press, 1978. 15. Baum, H., Dodgson, L. S., and Spencer, B., The assay of arylsulfatases A and B in human urine. Clin. Chim. Acta 4, 453-455 (1959). 16. Nicholls,R. G., and Roy, A. B., Arylsulfatases. In The Enzymes, 5, P. D. Boyel, Ed., Academic Press, New York, NY, 1971, pp 2141. 17. Dodgson, L. S., and Spencer, B., Studies on sulfatases.5. The determination of inorganic sulphate in the study of sulphatases. Biochem. J. 55, 436-440 (1953). 18. Bergmeyer, H. U., Gawehn, K., and Grasse, M., Enzymes as biochemical reagents. In Methods of Enzymatic Analysis, H. U. Bergmeyer, Ed., Academic Press, Inc., New York, NY, 1974, pp 452522. 19. Dzialosznski, L. M., Kroll, J. L., and Frohlich, A., Arylsulfatase activity of some malignant tumors. Clin. Chim. Acta 14, 450-453 (1966).
This investigation was supported in part by USPHS Grant CA16766 from the National Cancer Institute, through the National Large Bowel Cancer Project, and in part by USPHS Grant AH-00002-02. We gratefully acknowledge the assistance of J0 Ann Alleman and the Editorial Office of Louisiana State University Medical Center in New Orleans.
20. Geokas, M. C., and Rinderknecht, 13-glucuronidase in acute alcoholism. (1973).
References
22. Begum, A., and Ihyerah, T. R., Arylsulfatases and -glucuronidase activity in serum in kwashiorkor. Clin. Chim. Acta 28, 263-268 (1970).
1. Robinson,
D., Spencer,
B., and Williams,
R. T., Spectrophotometric
H., Plasma arylsulfatase and Clin. Chim. Acta 46, 27-32
21. Rinderknecht, H., Geokas, M. C., Carmack, C., and Haverback, B. J., The determination of arylsulfatases in biological fluids. Clin.
Chim. Acta 29, 481 (1970).
CLINICALCHEMISTRY,Vol. 25, No. 2, 1979 331
