- Email: [email protected]
SERUM-HEXOSAMINIDASE LEVELS IN CYSTIC FIBROSIS
1122
SERUM-HEXOSAMINIDASE LEVELS IN CYSTIC FIBROSIS SIR,-Several reports 1-3 have established the existence of metachromasia positive and negative varieties of cystic fibrosis (C.F.), as first described by Danes and Bearn,4 although metachromasia is by no means diagnostic of C.F. or its carrier state. The metachromatic status of a patient or carrier is determined by microscopic evaluation of metachromasia in cultured skin fibroblasts stained with toluidine blue O. The observation that at least two different phenotypic expressions of C.F. can be demonstrated, which appear to breed true in families, may be interpreted as evidence for the genetic heterogeneity of C.F. Several in-vitro studies of C.F. cells reported altered levels of various enzymes.5-7 We have studied total serumhexosaminidase (B-D-N-acetylglucosaminidase) as well as the levels of the A and B isozymes of hexosaminidase in C.F. homozygotes and heterozygotes previously characterised as metachromasia negative and positive. The metachromatic status of our C.F. serum donors was determined from their cultured skin fibroblasts by a method previously described.8Peripheral blood was drawn from 2 C.F. homozygotes and 1 heterozygote (parent of one of the homozygotes) determined to be metachromasia negative, and 1 C.F. homozygote and 3 C.F. heterozygotes (2 of these are the parents of the homozygote) determined to be metachromasia positive, Total serum levels of hexosaminidase, as well as the relative percentages of the A and B isozymes, were determined by the method of Okada and O’Brien 9:
In addition, after further convenient method for classifying individuals of either metachromatic status for future studies relevant to the heterogeneity of c.F. We do not feel that the hexosaminidase isozyme variation reported here is implicated in the aetiology of c.F., but rather represents a secondary effect of the mutant gene similar to those in previous reports.5-’ In this sense, these findings may be analogous to the raised levels of acid phosphatase in Gaucher’s disease." Since metachromasia is a characteristic of increased storage in lysosomes,’-1 it is probable that the metachromasiapositive form of C.F. represents either a lysosomal storage disease or enlargement of lysosomes due to the storage of an altered enzyme incapable of being secreted. The finding of elevated hexosaminidase A may be due to slow secretion of hexosaminidase, in which case most of the enzyme found in serum would represent the freshly secreted enzyme. Since hexosaminidase A is relatively unstable, it would make up a relatively larger fraction of the enzyme detected.
heterogeneity
of this disease.
verification, this study
may
serve as a
Division of Medical Genetics and Department of Pediatrics, Mount Sinai School of Medicine of the City University of New York, New York 10029, U.S.A.
JAMES H. CONOVER ELAINE J. CONOD KURT HIRSCHHORN.
MACROGLOBULINÆMIA OR MULTIPLE MYELOMA ?
SIR,-Your editorial (Feb. 7, p. 359) stressed that prominent feature of Waldenstrom’s macroglobulinsemia, hyperviscosity, could also be found in multiple myeloma. Lately, the distinction between the two diseases became less stringent, since many features considered characteristic for one disease were also found in the other. The following a
case
is relevant.
69-year-old man was admitted to hospital because of weakappetite, pallor, and impaired vision. He felt well until two weeks before admission and lost5 lb. (2-3 kg.) in these two weeks. He had tuberculosis a long time ago and had had diabetes mellitus for five years. He was very pale, and a few small mobile submandibular lymph-nodes were palpable. There were no other palpable nodes. The spleen and liver edge were palpable and the vertical span of the liver was 15 cm. The optic fundi showed dilated veins and hxmorrhages, more prominent in the left eye. The blood-pressure was 150/90 mm. Hg. The rest of the physical examination was normal. The urine was + + for protein and + glucose. The sediment contained a mixture of hyaline and granular casts. The heat test for Bence Jones protein was negative. The haemoglobin was 6-6 g. per 100 ml. and hxmatocrit 20%. There were 9300 leucocytes per c.mm. (28% segmented, 12% band-form neutrophils, 54% lymphocytes, 2% eosinophils, and 4% monocytes), rouleaux formation was present. The platelet-count was 82,000 per c.mm. and the reticulocyte count 0-1%. The erythrocyte A
ness, loss of *
Fluorometer units, Turner Fluorometer, model in, Palo Alto, California. t OH obligate heterozygote for cystic fibrosis. =
The normal control total serum level of hexosaminidase represents a mean value of more than 100 normal individuals studied in our laboratory over two years. The average normal relative percentages of isozymes A and B are those established by Okada and O’Brienand confirmed in our
laboratory. It can be seen that the metachromasia-negative donors exhibit generally higher total levels of enzyme, but the isozyme percentages are equivalent to the values established from our normal population, In contrast, the donors with metachromasia-positive status exhibit somewhat low total levels of enzyme activity, but the percentage of the B isozyme form is low, while that of the A isozyme is high, a situation which is the reverse of the carrier profile for TaySachs disease. Although this study is small and preliminary, it indicates a difference between two forms of c.F., distinguished by metachromasia, and thus is a further indication of the 1. 2.
Danes, B. S., Beam, A. G. J. exp. Med. 1969, 129, 775. Matalon, R., Dorfman, A. Biochem. biophys. Res. Commun. 1968, 33,
954. 3. Danes, B. 4. Danes, B.
5. 6.
7. 8. 9.
S., Flensborg, E. W. Am. J. hum. Genet. 1971, 23, 297. S., Beam, A. G. Lancet, 1968, i, 1061. Gibbs, G. E., Griffin, G. D. Science, 1970, 167, 993. Kraus, I., Antonowicz, I., Shah, H., Lazarus, H., Shwachman, H. Pediatrics, 1971, 47, 1010. Antonowicz, I., Sippell, W. G., Shwachman, H. Pediat. Res. 1972, 6, 803. Danes, B. S., Bearn, A. G. J. exp. Med. 1966, 123, 1. Okada, S., O’Brien, J. S. Science, 1969, 165, 698.
sedimentation-rate was 72 mm. in one hour. Creatinine 1-9 mg., uric acid 9-3 mg., glucose 287 mg., calcium 7’9 mg., phosphorus 4-2 mg., and cholesterol 84 mg. per 100 ml. Electrolytes and serum-enzyme activity were normal. The creatinine clearance was 54 ml. per hr. Serum contained 12 g. protein (2-5 g. albumin) per 100 ml. The prothrombin time was 14-2 sec. (control 11-2 sec.). X-rays of the bones were normal. The direct Coombs test was positive and the indirect test was negative. The Sia test was strongly positive at pH 7-2. The bone-marrow aspirate showed Serum electrophoresis remany plasmacytoid lymphocytes. vealed a homogeneous band in the slow gamma region. There were no cryoglobulins. While the diagnosis of macroglobulinxmia was considered, we were surprised to find that the serum relative viscosity was 2-4 (normal 14-1-8) and much decreased IgM and IgA were found on immunoelectrophoresis. Immunoglobulin G was present in a concentration of 6 g. per 100 ml. serum. By ultracentrifugation, a big increase in 7S and a decrease in 19S 10. Van Hoof, F., Hers, H. G. Eur. J. Biochem. 1968, 7, 34. 11. Gertner, M., Zalay, E., Hirschhorn, K. Clin. Genet. 1970,
1, 28.
SERUM-HEXOSAMINIDASE LEVELS IN CYSTIC FIBROSIS SIR,-Several reports 1-3 have established the existence of metachromasia positive and negative varieties of cystic fibrosis (C.F.), as first described by Danes and Bearn,4 although metachromasia is by no means diagnostic of C.F. or its carrier state. The metachromatic status of a patient or carrier is determined by microscopic evaluation of metachromasia in cultured skin fibroblasts stained with toluidine blue O. The observation that at least two different phenotypic expressions of C.F. can be demonstrated, which appear to breed true in families, may be interpreted as evidence for the genetic heterogeneity of C.F. Several in-vitro studies of C.F. cells reported altered levels of various enzymes.5-7 We have studied total serumhexosaminidase (B-D-N-acetylglucosaminidase) as well as the levels of the A and B isozymes of hexosaminidase in C.F. homozygotes and heterozygotes previously characterised as metachromasia negative and positive. The metachromatic status of our C.F. serum donors was determined from their cultured skin fibroblasts by a method previously described.8Peripheral blood was drawn from 2 C.F. homozygotes and 1 heterozygote (parent of one of the homozygotes) determined to be metachromasia negative, and 1 C.F. homozygote and 3 C.F. heterozygotes (2 of these are the parents of the homozygote) determined to be metachromasia positive, Total serum levels of hexosaminidase, as well as the relative percentages of the A and B isozymes, were determined by the method of Okada and O’Brien 9:
In addition, after further convenient method for classifying individuals of either metachromatic status for future studies relevant to the heterogeneity of c.F. We do not feel that the hexosaminidase isozyme variation reported here is implicated in the aetiology of c.F., but rather represents a secondary effect of the mutant gene similar to those in previous reports.5-’ In this sense, these findings may be analogous to the raised levels of acid phosphatase in Gaucher’s disease." Since metachromasia is a characteristic of increased storage in lysosomes,’-1 it is probable that the metachromasiapositive form of C.F. represents either a lysosomal storage disease or enlargement of lysosomes due to the storage of an altered enzyme incapable of being secreted. The finding of elevated hexosaminidase A may be due to slow secretion of hexosaminidase, in which case most of the enzyme found in serum would represent the freshly secreted enzyme. Since hexosaminidase A is relatively unstable, it would make up a relatively larger fraction of the enzyme detected.
heterogeneity
of this disease.
verification, this study
may
serve as a
Division of Medical Genetics and Department of Pediatrics, Mount Sinai School of Medicine of the City University of New York, New York 10029, U.S.A.
JAMES H. CONOVER ELAINE J. CONOD KURT HIRSCHHORN.
MACROGLOBULINÆMIA OR MULTIPLE MYELOMA ?
SIR,-Your editorial (Feb. 7, p. 359) stressed that prominent feature of Waldenstrom’s macroglobulinsemia, hyperviscosity, could also be found in multiple myeloma. Lately, the distinction between the two diseases became less stringent, since many features considered characteristic for one disease were also found in the other. The following a
case
is relevant.
69-year-old man was admitted to hospital because of weakappetite, pallor, and impaired vision. He felt well until two weeks before admission and lost5 lb. (2-3 kg.) in these two weeks. He had tuberculosis a long time ago and had had diabetes mellitus for five years. He was very pale, and a few small mobile submandibular lymph-nodes were palpable. There were no other palpable nodes. The spleen and liver edge were palpable and the vertical span of the liver was 15 cm. The optic fundi showed dilated veins and hxmorrhages, more prominent in the left eye. The blood-pressure was 150/90 mm. Hg. The rest of the physical examination was normal. The urine was + + for protein and + glucose. The sediment contained a mixture of hyaline and granular casts. The heat test for Bence Jones protein was negative. The haemoglobin was 6-6 g. per 100 ml. and hxmatocrit 20%. There were 9300 leucocytes per c.mm. (28% segmented, 12% band-form neutrophils, 54% lymphocytes, 2% eosinophils, and 4% monocytes), rouleaux formation was present. The platelet-count was 82,000 per c.mm. and the reticulocyte count 0-1%. The erythrocyte A
ness, loss of *
Fluorometer units, Turner Fluorometer, model in, Palo Alto, California. t OH obligate heterozygote for cystic fibrosis. =
The normal control total serum level of hexosaminidase represents a mean value of more than 100 normal individuals studied in our laboratory over two years. The average normal relative percentages of isozymes A and B are those established by Okada and O’Brienand confirmed in our
laboratory. It can be seen that the metachromasia-negative donors exhibit generally higher total levels of enzyme, but the isozyme percentages are equivalent to the values established from our normal population, In contrast, the donors with metachromasia-positive status exhibit somewhat low total levels of enzyme activity, but the percentage of the B isozyme form is low, while that of the A isozyme is high, a situation which is the reverse of the carrier profile for TaySachs disease. Although this study is small and preliminary, it indicates a difference between two forms of c.F., distinguished by metachromasia, and thus is a further indication of the 1. 2.
Danes, B. S., Beam, A. G. J. exp. Med. 1969, 129, 775. Matalon, R., Dorfman, A. Biochem. biophys. Res. Commun. 1968, 33,
954. 3. Danes, B. 4. Danes, B.
5. 6.
7. 8. 9.
S., Flensborg, E. W. Am. J. hum. Genet. 1971, 23, 297. S., Beam, A. G. Lancet, 1968, i, 1061. Gibbs, G. E., Griffin, G. D. Science, 1970, 167, 993. Kraus, I., Antonowicz, I., Shah, H., Lazarus, H., Shwachman, H. Pediatrics, 1971, 47, 1010. Antonowicz, I., Sippell, W. G., Shwachman, H. Pediat. Res. 1972, 6, 803. Danes, B. S., Bearn, A. G. J. exp. Med. 1966, 123, 1. Okada, S., O’Brien, J. S. Science, 1969, 165, 698.
sedimentation-rate was 72 mm. in one hour. Creatinine 1-9 mg., uric acid 9-3 mg., glucose 287 mg., calcium 7’9 mg., phosphorus 4-2 mg., and cholesterol 84 mg. per 100 ml. Electrolytes and serum-enzyme activity were normal. The creatinine clearance was 54 ml. per hr. Serum contained 12 g. protein (2-5 g. albumin) per 100 ml. The prothrombin time was 14-2 sec. (control 11-2 sec.). X-rays of the bones were normal. The direct Coombs test was positive and the indirect test was negative. The Sia test was strongly positive at pH 7-2. The bone-marrow aspirate showed Serum electrophoresis remany plasmacytoid lymphocytes. vealed a homogeneous band in the slow gamma region. There were no cryoglobulins. While the diagnosis of macroglobulinxmia was considered, we were surprised to find that the serum relative viscosity was 2-4 (normal 14-1-8) and much decreased IgM and IgA were found on immunoelectrophoresis. Immunoglobulin G was present in a concentration of 6 g. per 100 ml. serum. By ultracentrifugation, a big increase in 7S and a decrease in 19S 10. Van Hoof, F., Hers, H. G. Eur. J. Biochem. 1968, 7, 34. 11. Gertner, M., Zalay, E., Hirschhorn, K. Clin. Genet. 1970,
1, 28.