Measurements of arylsulfatases A and B in human serum

574

B r i e f clinical and laboratory observations

Measurements of arylsulfatases A and B in human serum ,lagat Singh, M.Sc., M.S., Ph.D., Daniela Tavella,* Ph.D., and Nicola Di Ferrante, M.D., Ph.D.,** Houston, Texas

been described recently for the m e a s u r e m e n t of various lysosomat e n z y m e s in h u m a n serum. ~-4 The possibility of performing these assays on serum samples is appealing because of the inherent ease, economy, arid rapidity of procuring samples and the immediacy of obtaining results. The methods proposed here are modifications of previously described ones for the measurement of arylsulfatases A and B in human urine s and of A R A in h u m a n serum. 4 They allow a rapid and accurate m e a s u r e m e n t of the two enzymes; the interference of A R A in the m e a s u r e m e n t of ARB was e l i m i n a t e d as p r e v i o u s l y recommended, s The methods described are valuable for the rapid diagnosis of metachromatic leukodystrophy, mucopolysaccharidosis VI (Maroteaux-Lamy disease), or a combination of the two diseases. 6 S E V E R A L M E T H O DS h a v e

MATERIALS

AND METHODS

A r e c e n t l y p u b l i s h e d m e t h o d 4 for arylsulfatase A assay could not be used as described because of formation of a heavy precipitate (probably due to Ca(OH) 2) upon addition of NaOH necessary for color development. Our proposed modification consists of an addition of m i n i m a l a m o u n t s of e t h y l e n e d i a m i n e t e t r a - a c e t i c acid to the reaction mixture to prevent formation of the precipitate. Two-tenths of a milliliter of substrate solution (containing 4 ixmoles of nitrocatechol sulfate i n pH 4.9, 1M From the Laboratory of Conneetive Tissue Research of the Marrs McLean Department of Biochemistry, from the Department of Medicine and the Division of Orthopedic Surgery of the Department of Surgery, Baylor College of Medicine. Supported in part by grants AM-10811, HL-05435, GM-19513, and GM-O0081-O1 of the United States Public Health Service by the National Foundation-March of Dimes, and by the Robert A. Welch Foundation. *On lea ve qf absence /?om the Departmentc~fPediatrics(f/he Catholic University, Rome, Italy. **Reprintaddress:Departmento/'Biochemistly, BaylorCollege of Medicine, Houston, Texas 77025.

The Journal of Pediatrics April 1975

sodium acetate buffer, 0.5 mM in Na4P207 and 1.7M in NaC1) and 0.2 ml of serum were incubated at 37~ for 4 hr: Thereafter, 0.08 ml of 2.5N NaOH and 0.1 ml of 0.15 mM EDTA in water were added to each tube, and the red color produced by the released nitrocatecbol was measured at 515 mix against water. Appropriate controis were made by incubating serum and substrate separately and combining them immediately prior to addition of NaOH and EDTA. The results reported in Table I are based on net absorbencies/hour, obtained by subtracting the values of the blanks from those of the respective reaction mixtures. Abbreviations used ARA: arylsulfatase A ARB: arylsulfatase B EDTA: ethylenediaminetetra-acetic acid N CS: nitrocatee hol sulfate The m e t h o d for arylsulfatase B assay follows the principles o u t l i n e d by Baum a n d associates s for the determination of the enzyme in the urine. However, using serum, it became necessary to eliminate the turbidity due to formation of Ba(OH) 2 upon addition of NaOH. In order to remove chloride ions, h u m a n serum was dialyzed at 5~ against a pH 6.0, 0.5M sodium acetate buffer, 10 mM in barium acetate. The first three changes of buffer were made every 2 hours, while the fourth dialysis period lasted overnight, and 1 liter of buffer was used for each change. In each tube, to 25 Ixmoles of NCS in 0.5 ml of water were added 0.3 ml of dialyzed serum and enough dialysis buffer to make a final volume of 1 ml. The tubes were incubated at 37~ after 2 and 4 hr, to each tube was added 0.5 ml of 3N NaOH, followed by 0.1 ml of 0.15 mM EDTA in water; the coloi produced was measured at 515 mix. Even in this assay, use was made of controls in which dialyzed serum and substrate were separately incubated; net absorbance was the difference between that of the incubation mixture and of the respective controls. The minor contribution that, under the condition of the assay, ARA makes to ARB activity was computed according to Baum and ass0ciates s by plotting the net absorbencies obtained at 2 and 4 hr against time and extrapolating the line to zero time. The value of the intercept (X), when multiplied by 0.2, gives 5an approximate measure of the activity to b e ascribed to ASA. The values for ARB reported in Table I represent corrected absorbence/hour, calculated according to the following expression: Corrected absorbence = [ A 4 - ( A 2 + 0.2 X)]/2, in

Volume 86 Number 4

Brief clinical and laboratory observations

575

T a b l e I. A r y l s u l f a t a s e B a n d a r y l s u l f a t a s e A activities in h u m a n s e r u m

Enzyme activities as:

Subject Normal S.J. S.J. S.J. H.F. N.Y. A.J. R.R. S.S. Average L.I. L.C. F.D. T.D. M.D. H.M. S.M. Average Maroteaux-Lamy patient L.D. R.C. Maroteaux-Lamy heterozygote R.M.

Sex M M M M M M M M F F F F F F F

Units* of ARB

Units* of ARA

t89.1 156.1~" 175.2 + 178.9 !07.1 71.5 201.5 146.3 153.2 + 44.1 49.6 147.3 80.6 60.1 61.9 188.5 146.3 104.9 + 54.8

667.4 592.6 542.7 327.1 403.0 320.9 249.7 215.6 414.9 ~+ 167.1 299.2 423.2 376.7 n.d. 366.4 217.6 386.7 345.0 _+ 74.4

M M

0.0 0.0

189.1 n.d.

F

313.1

367.4

Ralio: ARA/ARB 3.5 3.8 3.1 1.8 3.8 4,5 1.2 1.5 2,9 • 1.23 6.0 2.9 4.7 5.9 1.2 2.6 3.9 + 1.95

1.2

*1 unit of activity = 1 /,g of nitrocatechol (NC) released at 37~ C/br/dl of serum. The activity of ARB is based on the observed calibration coefficient of 125.6 nmole NC per optical density, and that of ARA on the calibration coefficient of 47.2 nmole NC per optical density. tActivity of the sample after storing frozen at --20 ~ for 2 days was 147.3 units. :~Activity of the sample after storing frozen at --20 ~ for 4 and 22 days were 175.5 units and 195.3 units, respectively. n.d. Not determined.

w h i c h A 2 a n d A 4 are a b s o r b e n c i e s at 2 a n d 4 hr, 0.2 is the c o n s t a n t p r e v i o u s l y suggested, X is t h e v a l u e o f t h e o r d i n a t e i n t e r c e p t , a n d 2 is t h e divisor in o r d e r to o b t a i n a b s o r b e n c i e s p e r hour.

RESULTS

AND DISCUSSION

The majority of determinations were performed on f r e s h sera, 0.4 ml b e i n g sufficient for t h e m e a s u r e m e n t of A R A a n d 1.2 m l for t h a t o f A R B . T h e sera c o u l d b e s t o r e d f r o z e n at --20 ~ for e x t e n d e d p e r i o d s o f t i m e witho u t a n y a p p a r e n t loss o f activities. It has b e e n previously r e p o r t e d that, w h e n p u r i f i e d p r e p a r a t i o n s o f s e r u m A R B are used, a p p r e c i a b l e loss o f activity o c c u r s d u r i n g l e n g t h y i n c u b a t i o n s a n d t h a t t h e a d d i t i o n o f cysteine to t h e i n c u b a t i o n m i x t u r e is able to p r e v e n t it. 7 W e h a v e n o t n o t i c e d a n y loss o f s e r u m A R B activity d u r i n g the v a r i o u s i n c u b a t i o n periods, p r o b a b l y b e c a u s e o f p r o t e c t i o n afforded t h e e n z y m e b y t h e p r o t e i n s p r e s e n t in s e r u m . T h e levels o f A R A a n d A R B f o u n d in sera o f normal males and females, of two patients with Maroteaux-Lamy syndrome, and of one obligate

h e t e r o z y g o t e for this disease are g i v e n in T a b l e I. T h e v a l u e s for A R A agree well with t h o s e o b t a i n e d b y o t h e r investigators who measured the enzyme activity on whole serum4or fractions thereof] N o r m a l m a l e s h a v e a s e r u m A R B activity h i g h e r t h a n t h a t o f n o r m a l f e m a l e s (153 _+ 44 v e r s u s 105 _+ 55 u n i t s ) ; t h i s finding, a n d t h e a b s o l u t e values o b t a i n e d w i t h the p r o p o s e d m e t h o d , are in a g r e e m e n t w i t h prev i o u s r e p o r t s 7 in w h i c h t h e e n z y m e was m e a s u r e d o n chromatographic fractions of serum. In agreement with recent contributions implicating A R B in t h e p a t h o g e n e s i s o f M a r o t e a u x - L a m y disease, 8-n t h e p r o p o s e d m e t h o d s h o w s a b s e n c e o f A R B activity in t h e sera o f two p a t i e n t s affected b y t h e disease. A s s a y s p e r f o r m e d o n h o m o g e n a t e s o f c u l t u r e d f i b r o b l a s t s a n d o n u r i n a r y p r o t e i n s of p a t i e n t L. D. also d e m o n s t r a t e d a b s e n c e o f A R B activity. 1~ Our l i m i t e d e x p e r i e n c e with o n e obligate heterozygote for M a r o t e a u x - L a m y trait fails to s h o w a n y differe n t level o f activity f r o m t h a t o f n o r m a l c o n t r o l subjects.

576

Brief clinical and 'laboratory observations

Several facts indicate that the procedure employed to correct ARB activity for any possible contribution due to A R A is valid also for serum assays: (1) the values for s e r u m ARB activity o b t a i n e d with this m e t h o d are comparable to those obtained for serum enzyme chromatographically separated from A R A 7 prior to assay; (2) patients with Maroteaux-Lamy syndrome who have a deficiency of ARB in homogenates of various organs, cultured fibroblasts, or in urinary protein precipitates have no ARB activity in serum, despite normal levels of serum A R A activity. Thus the methods described here remove a source of possible error in a previously described method for the m e a s u r e m e n t of A R A activity4 and allow an easy, rapid, and economic method for the diagnosis of MaroteauxLamy disease. REFERENCES 1. Singh J, Niebes P, and Di Ferrante N: A method for a-Liduronidase assay, FEBS Lett 45:248, 1974. 2. Glaser JH and Sly WS: /3-glucuronidase deficiency mucopolysaccharidosis: Methods for enzymatic diagnosis, J Lab Clin Med 82:969, 1973. 3. Beaudet AL, DiFerrante N, Ferry GD, Nichols BL Jr, and Mullins CE: Variation in the phenotypic expression of/3glucuronidase deficiency, J Pediatr 86:388, 1975.

Cardiovascular malformations associated with imperforate anus Ronald D. Greenwood, M.D., Amnon Rosenthal, M.D., and Alexander S. Nadas, M.D., Boston, Mass.

CARDIOVASCULAR MALFORMATIONS have b e e n reported to occur in 171 of 1,898 (9%; range 3.5-17%) i n f a n t s with imperforate anus. 1"1~A l t h o u g h coarctation,l,6,10 tetralogy of Fallot,3,6and other diagnoses have been made in isolated cases, the nature of the CVM and the prognosis of infants with IA and associated heart disease has not been defined. The present review of a large group of infants with IA seen during the last From the Department of Cardiology, The Children's Hospital Medical Center and the Department of Pediatrics, HarvardMedical School. Supported inpart by Training GrantNo. HL05855, ProgramProjectHL10436, from The National Institutes of Health.

The Journal of Pediatrics April 1975

4. Beratis NG, Aron AM, and Hirschhorn K: Metachromatic leukodystrophy: Detection in serum, J Pediatr 83:824, 1973. 5. Baum H, Dodgson KS, and Spencer B: The assay of arylsulfatases A and B in human urine, Clin Chim Acta 4:453, 1959. 6. Moser HW: Sulfatide lipidosis: Metachromatic leucodystrophy, in Stanbury JB, Wyngaarden JB, and Fredrickson DS, editors: The metabolic basis Of inherited disease, New York, 1972, McGraw-Hill Book Company, Inc. p 688. 7. Gniot-Szulzycka J: Arylsulfatases A and B in blood, Clin Chim Acta 32:17, 1971. 8. Kihara H, Fluharty AL, and Stevens RL: Arylsulfatase B deficiency in Maroteaux-Lamy fibroblasts, Am J Hum Gen 25:41A, 1973. 9. Stumpf DA, and Austin JH: Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome). I. Sulfatase B deficiency in tissues, Am J Dis Child 126:747, 1973. 10. Di Ferrante N, Hyman BH, Klish W, Donnelly PV, Nichols BL, Dutton RV, and Gniot-Szulzycka J: Mucopolysaccharidosis VI (Maroteaux-Lamy disease): Clinical and biochemical study of a mild variant case, Hopkins Med J 135:42, 1974. 11. Di Ferrante N, Singh J, Donnelly PV, and GniotSzulzycka J: Maroteaux-Lamy mucopolysaccharidosis (type VI) and arylsulfatase B deficiency, Fed Proc 33:1520, 1974.

decade at our hospital addresses itself to these problems. METHODS The diagnostic files in the Medical Records Departm e n t of the C h i l d r e n ' s Hospital Medical C e n t e r , Boston, were searched and the records of all patients with IA from 1962 through 1973 were reviewed. Included were all patients fulfilling the criteria proposed Abbreviations used CVM: Cardiovascular malformations IA: imperforate anus by Gross and Ladd for IA. 1~ Excluded were patients with ectopic anus or higher intestinal atresias. In infants with any evidence of cardiac disease, the chest roentgenogram, electrocardiogram, cardiac catheterization data, and postmortem findings (when available) were reviewed. In addition, the diagnostic files of the New E n g l a n d Regional I n f a n t Cardiac Program were searched for infants admitted between 1968 and 1972 with an associated diagnosis of IA. The Regional I n f a n t Cardiac Program includes all infants born in the six