Clin. Biochem. 14 (3) 177-181 (1981)

Comparison of the Tartrate-Resistant Acid Phosphatase in Gaucher's Disease and

Leukemic Reticuloendotheliosis* KWOK-WAI LAM, ~ CHIN-YANG LI, 2 LUNG T. YAM, ~ and ROBERT J. DESNICK 4

Departments of Ophthalmology and Biochemistry, Albany Medical College Albany, N.Y. 12208.

(Accepted July 15, 1981)

Tartrate-resistant acid phosphatase was isolated from serum and spleen of patients affected by Gaucher's disease. Elec- trophoretic and antigenic properties were compared to the en- zyme isolated from hairy cells described in a previous study (9). The enzyme isolated from Gaucher serum has electrophoretic and antigenic properties identical to the acid phosphatase band 5b of hairy cells. The major tartrate-resistant acid phosphatase in the Gaucher spleen is band 5a. Bands 5a and 5b have identical protein structure indicated by their identical antigenicity. The removal of carbohydrate from band 5a by sialidase converted band 5a to 5b.

THE EARLIER STUDIES OF ACID PHOSPHATASE ISOEN- ZYMES among normal and leukemic cells led us to recognize the clinical significance of the tar t ra te- res is tant acid phosphatase (isoenzyme 5b) as a specific marker for the histochemical detection of ret iculum cells in leukemic ret iculoendotheliosis (hairy cell leukemia) (1). The fast electrophoretic mobility toward the cathode on acidic acrylamide gel and s t rong affinity to CM-cellulose are special characteristics of thie en- zyme and distinguish it from the acid phosphatase iso- enzymes in normal tissues (2). The enzyme is compart- mentalized inside the subcellular granules of the hairy cells, and does not leak into the plasma (3). Leakage of t a r t ra te res i s tan t acid phosphatase from pathologic tissues into the circulation has been observed in malignancies metastast ized to bone and in Gaucher's disease (4, 5, 6). A recent report showed that the eleva- t ion of se rum ta r t r a te - res i s t an t acid phosphatase assoc ia ted wi th ma l ignanc ies was der ived from osteoclasts (7, 8). The isoenzyme 5 of hairy cells and that of osteoclastic bone tumors have similar biochemical and an t igen ic proper t ies (9). The p re sen t repor t describes the multiplicity of ta r t ra te- res is tant acid phosphatase in the spleen and serum of pat ients with Gaucher's disease and their relationship to the enzyme in hairy cells.

1Author to whom correspondence should be sent. 2Department of Laboratory Medicine and Surgical Pathology, Mayo Clinics, Rochester, MN 55901. aDivision of Hematology and Oncology, Veterans Administra- tion Hospital, Louisville, KY 40202. 4Division of Medical Genetics, The Mount Sinai Medical Center, New York, NY 10029.

*This article was contributed by the National Academy of Clinical Biochemistry (NACB), through Dr. M. Rafelson.

MATERIALS AND METHODS

1. Specimen preparation:

Serum was obtained from six patients ranging in age from 3- 50 years with Type 1 Gaucher's disease. Three had undergone splenectomy. For these studies, the acid phospbatase was isolated from 50 ml of serum from a 25-year old female with Type 1 Gaucher's disease who bad been splenectomized at age 8 years. The acidity of the serum was adjusted to pH 5 with acetic acid, and subjected to centrifugation at 20,000 g for 15 rain. The supernatant fraction was applied to a CM-cellulose column (1.5 x 30 cm, equilibrated in 10 mmolar citrate buffer, pH 5.0), and eluted by a linear concentration gradient of NaC1. The salt concentration increased from 0.05 M to 1 M in 200 ml of citrate buffer (10 mmolar, pH 5).

The spleen from a patient with Type 1 Gaucher's disease, removed at surgery, was stored at -70 ° C until use. The frozen specimen was homoganized (lg/ml saline) in an Osterizer (max- imal speed). The homogenate was subjected to freeze-thaw 3

. . . . . " : .~ ~ ~ ~".~:~ Z ( ~ : ,

Fig. I -- Electrophoresis of tartrate-resistant acid phosphatase in serum of Gaucher's disease: A. lOpl of serum of Gaueher's disease. B. Mixture of 10 ~l of A with 0.5 m U of isoenzyme 5b purified

from hairy ceil C. 0.5 mU of isoenzyme 5.

178 LAM, LI, YAM AND DESNICK

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Fig. 2 -- CM-ceUulose chromatography of Gaucher serum: Serum specimen of Gaucher's disease was absorbed on the col- umn and eluted by NaCl as described in the text. Only one ma- jor activi ty peak was observed.

Gradient star t 80 mU 5b from 60 mU 5b from

Gaucher serum Hairy Cel ls

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./.

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./, -o.~ M 30 mU 5a from 20 mU 5b from ..'• , Gaucher spleen Hairy Cells

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Fig. 4 -- Ac id phosphatase in normal and pathologic human splee~ Saline extract of a normal human spleen {autopsy), the spleen affected by leukemic reticuloendotheliosis and that by Gaucher's disease were subjected to electrophoresis in duplicate. One gel column was stained for acid phosphatase and the other stained in the presence of 50 m M tartrate. The normal spleen contains bands 3 and ~ {.4). No activi ty band was seen in the normal spleen when tartrate was added. The hairy cell spleen contained bands 3, ~ and 5b (C). Tartrate eliminates bands 3 and 4, leaving 5b unaffected {1)). The Gaucher spleen /E) contains 3, 4, 5a and 5b. The tartrate inhibits bands 3 and 5, while 5a and 5b were unaffectec£

An ti body Fig. 3 -- Ant igenic ident i ty of the acid phosphatase in the serum of Gaucher's disease and isoenzyme 5b of hairy celt The enzyme isolated from serum (see Figure 2J was placed in the upper left and isoenzyme 5b of hairy cell was placed in the up- per right. An t i serum was placed in the lower welL Identical precipitin band was observed.

times, then subjected to centrifugation at 20,000 g for 20 rain. The supernatant fraction was dialysed overnight against 10 mmolar acetate buffer, pH 5.0, and subjected to centrifugation again. The clear supernatant fraction was applied to a CM- cellulose column (1.5 x 30 cm) and eluted by NaCl as described above.

Acid phosphatase, band 5b from hairy cells and the specific antiserum were prepared as previously described (9).

2. Colorimetric analyses of acid phosphatase:

P-nitrophenyl phosphate was used as the substrate for quan- titative analyses of acid phosphatase activity. The substrate concentration was 4 mmol/L. At the end of ten minutes incuba- tion at 37°C, the reaction was stopped by the addition of 2 ml of 0.1 N Na0H. The absorbance of the solution was measured at 410 nm. One mU is defined as nmoles substrate hydrolyzed per minute under the assay conditions described above.

3. Electrophoresis:

Acid phosphatase isoenzymes were analyzed by elec- trophoresis on acidic acrylamide gel. After 75 minutes of elec- trophoresis (5 ma. per sample), the activity bands were stained for 1 hour, using 1-naphthyl phosphate-Fast Garnet GBC as previously described (10).

4. Immunodiffusion.

lO/o agarose was dissolved in 0.15 mol/L NaCI. Sample wells were cut at a distance of 7 mm from the central well. Diffusion was allowed to occur overnight in a humid chamber at room temperature. The agarose slide was immersed in 250 ml of 0.15

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TARTRATE-RESISTANT ACID PHOSPHATASE IN GAUCHER'S DISEASE

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Band 5b

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Fig. 5 -- CM-cellulose chromatography of Gaucher spleen" The spleen extract was absorbed on the column and eluted by NaCl as described in the text. Al iquots of O.01 ml f rom each fraction were ana lyzed f o r acid phosphatase ac t i v i t y us ing p-nitrophenyl phosphate as substrate.

mol/L NaC1 for overnight, then stained for acid phosphatase ac- tivity using 1-naphthyl phosphate and Fast Garnet GBC as previously described (9).

RESULTS

The major acid phosphatase in the sera of the Gaucher patient was band 5b, which was identical to the tartrate-resistant acid phosphatase isolated form hairy cells (Figs. 1A and C). The identical electrophoretic mobility of the enzyme isolated from the two sources was confirmed by mixing the purified isoenzyme 5b with the serum from Gaucher's disease (Fig. 1). Only one dominant band {Fig. 1, B) was seen in the mixed sample.

The acid phosphatase in the serum was isolated by

179

CM-cellulose column chromatography; only one major peak was observed (Fig. 2). The enzyme isolated from Gaucher serum was identical antigenically to the en- zyme isolated from hairy cells (Fig. 3).

The acid phosphatase isoenzymes in the Gaucher spleen were compared to that of a normal spleen (autop- sy) and a spleen from a patient with hairy cell leukemia (Fig. 4). The major acid phosphatase forms in the normal spleen were bands 3 and 4 (Fig. 4A), which were com- pletely inhibited by t a r t r a t e (Fig. 4B). The characteristic of the spleen from hairy cell leukemia was the strong band 5b (Figs. 4C and 4D) which was the only band resistant to tartrate inhibition (Fig. 4D). The characteristic of the Gaueher spleen was the strong band 5a and a small amount of band 5b (Fig. 4E). Both bands 5a and 5b were resistant to tartrate inhibition (Fig. 4F).

The acid phosphatases in the Gaucher spleen were separated on a CM-cellulose column into multiple peaks (Fig. 5) and chromatographic fractions were subjected to polyacrylamide gel electrophoresis (Fig. 6). The first peak often contained bands 3 and 5a as shown in Fig. 6 (fraction 9). The front of the second contained mainly band 3. Band 4 appeared at the end of the second peak. Peak III was the major peak detected by the col- orimetric method, but had no detectable activity band when it was analyzed by electrophoresis. Peak 4 con- tained band 5a and followed by a small amount of band 5b.

The molecular weight of band 5a isolated from Gaucher spleen was compared to that of band 5b isolated from hairy cells by high pressure liquid chromatography. The ability of the column to separate proteins according to their molecular weight is il- lustrated in Fig. 7. The molecular weights of albumin, hemoglobin and cytochrome c are 69,000, 32,000 and 13,000 respectively. The separation of the above pro- teins by high pressure liquid chromatography is shown in Fig. 7A. The molecular weights of bands 5a and 5b was indistinguishable by high pressure liquid chromatography (Fig. 7) in spite of the distinct separa- tion by electrophoresis on acrylamide gel. Both bands 5a and 5b formed a line of identity with the antiserum prepared for band 5b of hairy cells (Fig. 3).

Sialidase had no effect on the electrophoretic mobili- ty of band 5b, yet it converted band 5a to a form which migrated with 5b (Fig. 8). After ten minutes of incuba- tion of 5a with sialidase, a significant amount of 5b was observed (Fig. 8B). After one hour of incubation, a strong band 5b was observed (Fig. 8C). The quantity of sialidase used in this study had no effect on the elec-

3 ~ ~ i ~ ¸̧ :̧ :̧ : 4 " 4 :

5a 5b

m

Fig. 6 -- Electrophoretic properties of acid phosphatase isoenzymes separated on CM-cellulose: Al iquots of 0.05 ml in each fraction separated on CM-ceUu- lose as shown in Figure 5 were sub- jec ted to electrophoresis and stained for acid phasphatase, using 1-naphthyl phosphate as substrate. The fraction numbers were indicated in the column.

9 11 13 15 17 19 21 38 42 44 47 48 51

180 LAM, LI, YAM AND DESNICK

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B

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20 40 60 80 I00 120

MINUTES

Fig. 7 -- Molecular weight determination by high pressure li- quid chromatography: TSK-gel column type G 3000 was equilibrated in O. 15 M NaCl and eluted at O. 5 ml per mira [A] a mixture of 30 #g cytochrome c fm.w. 13,000), 20 #g hemoglobin ?m.w. 32,000] and 30 #g albumin Im.w. 6&O00]. (BJ 100 #g albumin mixed with 160 m U isoenzyme 5~ fC/100 ~g albumin mixed wi th 100 m U isoenzyme 5b.

A B C D

Fig. 8 -- Ef fec t of sialidase on isoenzyme 5~" fA] 0.5 m U isoen- zyme 5o. ?BJ 0.5 m U isoenzyme 5a incubated with 200 m U sialidase (Sigma, Type IX) for ten minutes and ?CJ 0.5 m U isoenzyme 5a incubated with 200 m U sialidase for 60 mira (D] 0.5 m U isoenzyme 5b. The sialdase preparation contains < 0.005 mUpro tease activity.

trophoretic mobility of band 4, 5b, albumin and hemoglobin. The negative results of sialidase on the other proteins indicate that the sialidase did not contain sufficient protease to cause non-specific alteration of all proteins.

DISCUSSION

Elevation of tartrate-resistant acid phosphatase in the serum of patients with Gaucher's disease was first observed by Tucbman (6). Electrophoretic analyses of acid phosphatase in the sera from patients with Gaucher's disease showed a Strong band 5, similar to the tartrate resistant acid phosphatase of hairy cells (1). An unique characteristic of the tartrate-resistant acid phosphatase 5 in hairy cells is the high affinity of isoen- zyme 5 for CM-cellulose (2). A salt concentration of 0.4 M in the elution buffer is necessary to elute isoenzyme 5 from the cation exchange resin. Mercer et al. (11) ex- ploited the cationic property of the enzyme to design a mieroeolumn method for the detection of tartrate- resistant acid phosphatase 5 in Gaucher serum. They observed one peak of t a r t r a te - res i s t an t acid phosphatase in the serum of Gaucher's disease as the elution buffer was increased above 0.4 M NaCI. Using the microcolumn method, Robinson and Glew(12) observed two acid phosphatase peaks from an extract of Gaueher spleen. A tartrate-sensitive isoenzyme was eluted from the microcolumn in the same position as isoenzyme 5 of hairy cells. The resolution of acid phosphatase isoenzymes by the microcolumn method did not distinguish bands 5a and 5b.

In contrast, ion-exchange column chromatography was used in this study as the first step to resolve the multiple acid phosphatase in serum and in the spleen from patients with Gaucher's disease. Each acid phosphatase peak eluted from the column was examin- ed by acidic acrylamide gel electrophoresis. The pre- sent data showed a distinct difference between the isoenzyme 5 patterns in the serum and spleen from Gaucher's disease. In Gaucher serum only band 5b was detected while the major isoenzyme in Gaucher spleen was band 5a. Both had molecular weights very similar to hemoglobin (dimer molecular weight of 32,000 (12)) by high pressure liquid chromatography and were an- tigenically identical. The similar molecular weights, the conversion of band 5a to band 5b by sialidase and the identical antigenicity indicated that the two bands (Sa and 5b) had identical protein structures, but different carbohydrate content.

On the basis of physical, enzymatic and immunologic studies, it appears that the tartrate-resistant isoen- zyme 5 occurs as two forms, one sialylated (5a} and the other containing little, if any, sialic acid residues (5b). The desialylated form (5b) was the major tartrate- resistant acid phosphatase detected in Gaucher sera. This in contrast to the finding of Ashwell and Morell (14) and others that most circulating glycopro- teins are sialylated, and that sialidase treatment results in the rapid plasma clearance and receptor-mediated up- take of these proteins by hepatocytes. Since the major form in Gaucher spleen is 5a, and since non- splenectomized and splenectomized patients have 5b in

TARTRATE-RESISTANT ACID PHOSPHATASE IN GAUCHER'S DISEASE 181

their sera, it is unlikely that splenic cells are the source of the circulating 5b isoenzyme. In fact, the levels of serum 5b were high in splectomized patients. I t is in- t r iguing to speculate that the 5b form may represent cell specific isoenzyme or an incompletely post- t ranslat ionally modified form released from the tur- nover of Gaucher cells and/or osteoclasts in the marrow of pat ients with this disease. If this proves to be the case, the level of 5b in the serum may be correlated with the severi ty or activity of bony disease in these pa- t ients.

ACKNOWLEDGEMENT

The authors are grateful for the technical assistance of Mrs. Linda Lai. This work was supported in par t by PHS Research Grand CA 31187, awarded by the Na- tional Cancer Inst i tute , DHHS, PHS Research Grant GM 29862, GM 25279 awarded by the National Ins t i tu te of General Medical Science, a grant (1-438) from the March of Dimes Birth Defect foundation, and by the Veterans Adminis t ra t ion Hospital.

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2. Lain, K.W. and Yam, L.T. Biochemical characterization of the tartrate resistant acid phosphatase of human spleen with leukemic reticuloendotheliosis as a pyrophosphatase. Cli~ Chem. 23, 89-94 (1977).

3. Lam, K.W., Dekker, P.T., Castleman, J. and Yam, L.T. Studies of intracellular distribution of acid phosphatase 5

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4. Li, C.Y., Chuda, R.A., Lam, K.W. and Yam, L.T. Acid phosphatases in human plasma. J. Lab & Cli~ Med~ 82, 446-460 (1973).

5. Yam, L.T. Clinical significance of the human acid phosphatases. Am. J. Me& 56, 604-616 (1974).

6. Tuchman, L.R.0 Suna, H., and Carr, J.J. Elevation of serum acid phosphatase in Gaucher's disease. J. Mr. Sinai Hosp. 23, 227-229, (1956).

7. Lam, K.W., Eastlund, T., Li, C.Y. and Yam, L.T. Biochemical properties of tartrate-resistant acid phosphatase in serum of adults and children. Clin. Chem. 24, 1105-1108 (1978}.

8. Chen, J., Yam, L.T., Janckila, A.J., Li, C.Y. and Lam, K.W. Significance of "high" acid phosphatase activity in the serum of adults and children. Cli~ Chem. 25, 719-722 (1979).

9. Lain, K.W., Lee, P., Li, C.Y. and Yam, L.T. Immunological and biochemical evidence for identity of tartrate-resistant isoenzymes of acid phosphatases from human serum and tissues. Clin. Chem. 26, 420-422 (1980).

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11. Mercer, D.W., Peters, S.P., Glew, R.H., Lee, R.E., Wenger, D.M. Acid phosphatase isoenzymes in Gaucher's disease. Clin. Chem. 3, 631-635 (1977).

12. Robinson, D.B. and Glew, R.H. A tartrate-resistant acid phosphatase from Gaucher spleen. Purification and Pro- perties. J. BioL Chem. 255, 5864-5870 (1980).

13. Edelstein, S.J., Rehman, M.J., Olson, J.S. and Gibson, Q.H. Functional aspects of the subunit association- dissociation equilibrium of hemoglobin. J. BioL Chem. 245, 4372-4381 (1970).

14. Ashwell, G. and Morell, A.G. The role of surface car- bohydrate in the hepatic recognition of circulating glycoproteins. In: Advanced Enzymology, Vol. 41. A. Meister (ed) John Wiley, New York, 1974, pp. 99-128.

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