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Elevated serum and spleen angiotensin converting enzyme and serum lysozyme in Gaucher's disease
21
Clinica Chimica Acta, 74 (1977) 21-25 0 Elsevier/North-Holland Biomedical Press
CCA 8138
ELEVATED
SERUM AND SPLEEN ANGIOTENSIN
ENZYME
AND SERUM LYSOZYME
EMANUEL
SILVERSTEIN
CONVERTING DISEASE
* and JOAN FRIEDLAND
Laboratory of Molecular Biology, Department Biochemistry, State University of New York, N.Y. 11203 (U.S.A.)
(Received
IN GAUCHER’S
of Medicine and Graduate Downstate Medical Center,
Program in Brooklyn,
June 4th, 1976)
Summary In adult chronic non-neuronopathic (Type 1) Gaucher’s disease significant (p < 0.001) elevations of angiotensin converting enzyme in serum (93.3 f 14.8 nmol/min/ml; number elevated, 8/11; normal control 32.2 + 1.30, n = 58) and spleen (5.62 f 0.35 nmol/min/mg protein, n = 2; control, 0.431 + 0.101, n = 4) and serum lysozyme (15.6 f 3.37 pg/ml; number elevated, 4/5) were observed. The KM for hippuryl-L-histidyl-L-leucine of Gaucher (1.31 mM) and normal (1.23 mM) serum angiotensin converting enzyme were similar. The increased angiotensin converting enzyme (ACE) in Gaucher’s disease may be related to the genetic defect resulting in increased ACE synthesis in Gaucher cells, or perhaps generally, while high lysozyme may reflect an increased body mass of reticuloendothelial cells. These enzyme elevations may be of use in suggesting the possible presence of Gaucher’s disease and perhaps in assessing the magnitude of pathologic involvement.
Introduction Angiotensin converting enzyme (ACE) and lysozyme are elevated and positively correlated in the serum [l-6] and granulomatous lymph nodes [3,6--81 in sarcoidosis, perhaps due to active synthesis by the sarcoid granulomas which contain epithelioid cells of the reticuloendothelial series. While ACE was not elevated in rat, mouse and rabbit macrophages, rat Freund’s adjuvant granulomas [9] and in human blood leukocytes and lymph nodes in a variety of diseases [ 81, we have found striking elevation of serum and spleen ACE and serum lysozyme in Gaucher’s disease which is characterized by massive accumulation of large characteristic reticuloendothelial cells (Gaucher’s cells) laden with glu* To
whom
correspondence
should
be addressed.
cocerebroside due to deficient glucocerebroside-/3-glucosidase [ 10,111. An abstract of this work [ 121, a brief paper on elevated ACE [ 131 and an abstract on elevated lysozyme [ 141 in Gauchcr’s disease have been reported. Materials and methods ACE was assayed fluorimetrically with the substrate hippuryl-L-histidyl-Lleucine [ 151. Lysozyme was determined turbidimetrically with the substrate Micrococcus lysodeihticus [16] and is given in terms of micrograms hen egg white lysozyme equivalents. Sera were obtained from blood samples which were allowed to clot for about 1 h at room temperature and were stored at -85°C until use. The preparation of tissue extracts for lysozyme assay is described elsewhere [ 161. Tissues for ACE assay were fractionated at 0--5°C from 5% homogenates in 50 mM potassium phosphate, pH 8.3, prepared by 40 seconds (total) homogenization in a tissue homogenizer. The 254 X g (10 min centrifugation) and 39 000 X g (1 h centrifugation) pellets were washed once by centrifugation and resuspended to the original homogenate volume by brief homogenization. The two pellets and the final supernatant were assayed for ACE. The diagnosis of Gaucher’s disease was made in all cases by the finding of Gaucher cells in tissue specimens, as well as by determination of diminished tissue P-glucosidase in most cases. Results and discussion The mean serum ACE in 11 patients with Gaucher’s disease was significantly elevated about 3 fold (Table I) as compared to normal subjects (p < 0.001). Two of the three subjects with serum ACE in the normal range were asymptomatic, while the third had severe disease and was the only subject on steroid therapy. Perhaps steroid therapy suppressed an elevated serum ACE level in the third patient, since steroid therapy usually suppresses elevated serum ACE levels in sarcoidosis [ 171. ACE in two Gaucher spleens was significantly elevated about 13 fold compared to control tissues (p < 0.001). Most of the ACE activity was particulate in both Gaucher and control spleens (Table II). Numerous Gaucher cells were present in the Gaucher spleens. No blood leukocyte ACE activity was detected in 2 patients with normal serum ACE. The Klvr for hippurylL-histidyl-L-leucine of serum ACE from a Gaucher’s disease patient with elevated serum ACE (1.31 mM) was similar to that of normal serum ACE (1.23 mM). Serum lysozyme was elevated in 4 of 5 patients studied (Table I). Lysozyme generally paralleled ACE and was normal in a patient with a low normal ACE. Lysozyme in two spleens from subjects with Gaucher’s disease was in the midrange of values for control spleens (Table III), which normally contain abundant lysozyme-rich reticuloendothelial cells. The serum ACE results in nine adult patients and spleen ACE values in one Gaucher patient and four controls which have recently been reported [ 131 are in general agreement with results here given, although the Gaucher spleen ACE was about half that found in our two cases, and the reported KM for hippuryl-
23 TABLE
I
SERUM
ANGIOTENSIN
Patient
* -
NO.
Age
Sex
1
42
F
2 3 4 5
12 29 50 45
n9
6
CONVERTfNG
ACE (nmol/min/ml)
ENZYME
AND LYSOZYME
Clinical description
Lysozyme
5.5
44.1 59.5 84.0 178
14.5
M M M
63
M
105
7 8 9 10
70 48 65 48
F F F M
31.0 145 138 97.6
10.8 29.3 13.0
11
18
F
107
20.0
32.2 t 1.30 (n = 58) [151 12.5-51.9 93.3 i 14.8
fi.4 (0 = 50) [I81 O-l 0 15.5 + 3.37
8111
5f6
* All patients
were white except
DISEASE
Qg/mU
26.2
Normal mean !z S.E.M Normal range Mean Gauchar’s disease Number Elevated
IN GAUCHER’S
Progressive hepatosplenomegaly. bone pain: on steroids AsymBtomatic: icteric: past splenectomy Asymptotic: minimal splenomegaly Asymptotic: marked throbocytopenia; splenomegaiy Moderate bone disease: chronic renal disease on dialysis: splenectomv Substantial bone disease, anemia. pulmonary emphysema, spfenectomy ASympt#matiC Severe, bone involvement; anemia Splenomegaly; benign monoclonal gammopathv Moderate splenomegaly, no Gaucher’s disease symptams: recent nephrotic syndrome Symptomntic. splenectomy
No. 5 who was black.
L-histidine-b-leucine was about 10.fold lower than we have found for human serum and 17-fold lower than the rabbit lung ACE value [ 19,201. The LO-fold lower control spleen lysozyme as compared to Gaucher spleen lysozyme which has been reported [ 141 contrasts with equal values in this study. Since we have observed [ 161 that extraction of the strongly basic molecule lysozyme from various tissues at neutral pH was strongly dependent on TABLE
II
ELEVATION SEASE
OF
Condition
Gaucher’s Gaucher’s
ANGIOTENSIN
Angiotensin
disease disease
Normal Normal Diabetes Tuberculosis
converting
ENZYME
enzyme
254X8 pellet
39 000 x &! pellet
8.09 3.47 0.52 0.32 0.88
Gaucher f. S.E.M @ < 0.0011 Mean control * S.E.M. Mean
CONVERTING
IN THE
SPLEEN
IN GAUCHEK’S
~~rnol~min~mg protein)
_I-
Supernatant
Total
23.9 30.0
2.26 0.55
5.27 5.97
3.06 2.12 1.17 4.00
0.20 0.02 0.06
0.68 0.32 0.22 0.50
_-
5.62 + 0.350 0.43 i 0.101 -1-
DI-
TABLE
III
SPLEEN ._
LYSOZYME
IN GAUCIIER’S
Condition
DISEASE
Lysozyme
Gaucher’s
disease
1
4.94
Gaucher’s
disease
2
5.17
Mean
Gaucher’s
disease
5.06
Normal
8.01
Diabetes
4.30
Bronchopneumonia
1.21
NOrmaI
6.46
Tuberculosis
5.51
Mean
5.10
control
AND
(I.rg/mg
CONTROLS
protein)
+ 0.115
i
1.14
ionic strength, 1 M NaCl was used for lysozyme extraction in the present study. We have found that about 2-3s of Gaucher spleen lysozyme extractable with 1 M NaCl is extractable in distilled water in a 5% homogenate in contrast to virtually no extraction of control spleen lysozyme, probably accounting for the reported elevated Gaucher spleen lysozyme [ 141. Increased serum lysozyme may result from synthesis by an increased body mass of reticuloendothelial cells [ 211. Increased ACE may be related to the genetic defect in Gauche& disease which may result in increased synthesis of ACE generally or perhaps specifically in Gaucher’s cells, perhaps analogous to the mechanism hypothesized for sarcoidosis [ 221. These increased serum enzymes, particularly ACE, may have diagnostic value in Gaucher’s disease and the level of increase may reflect the magnitude of disease and the severity of the defect. The relationship of the elevation of ACE, which is not a lysozomal enzyme, to that of various acid hydrolases [23] and transcobalamin II [ 241 is not clear. Acknowledgments We are indebted to Dr. Neal Weinreb, Dr. Arthur Sawitsky, Dr. Irving Swiller, Dr. Stanley Lee, Mrs. Phyllis Schwartz and Mrs. Fran Berkwits for Gaucher serum samples, Dr. Arthur Sawitsky for Gaucher spleen samples, and Dr. Yale Rosen for histologic sections. We thank Charlotte Setton for able technical assistance. References Lieberman,
J.(1975)
Silverstein,
E.,
Silverstein, and
I?.,
Other
Friedland, Pascual,
Am.
Friedland,
J. Med. J..
Friedland,
J.,
Granulomatous Gee.
J.B.L.
Lyons.
Disorders.
J. and Silverstein, R.S.,
59,
Lyons,
E. (1976) and Finch.
Silverstein.
E..
Friedland,
J. and
Silverstein,
E.,
Friedland.
J., Lyons,
Silverstein,
E..
2137-2141
Friedland.
J..
365-372 H. and Kitt, H.A.
and
Ann.
N.Y. (1973)
H. and H.A.
T.
(1975)
Acad.
Biochem. S.C.
Ackerman,
Lyons.
M.
Gourin,
and
278.
Med.
15.
178-185
New.
Eng. Clin.
A.
Gourin,
Res.
(1976)
Sci.,
(1975)
Gourin,
Clin. A.
(1975) A.
352A Int.
Conf.
on
Sarcoidosis
498-513
J. Med. Res.
23.
Seventh
23,
Clin. (1976)
289,
1074-1076
600A Res. Proc.
23.
352A
Natl.
Acad.
Sci.
U.S.A.
73,
25
9 10
Silverstein,
E. and
Fredrickson.
D.S.
Wyngaarden,
J.B.
11
Brady,
12
Silverstein,
R.O.,
Friedland, and and
Kanfer,
J. (1976)
Sloan,
H.R.
Fredrickson. J.N.
and
E. and
Friedland,
J. and
Beutler.
Fed.
D.S.,
Shapiro. J. (1976)
E. (1976)
Biochem.
15
Friedland,
16
Silverstein,
E.,
Friedland.
J. and Ackerman,
17
Silverstein.
E.,
Friedland.
J., Lyons.
18
Prockop.
19
Cushman,
20
Das,
Res.
J. and Silverstein,
D.J.
and
D.W.
W.D.
and Cheung,
H.S.
S..
Z.A.
23
Ockerman,
P.A.
24
Gilbert,
H.S.
Todd,
Cohn,
E. (1976)
Med.
and Kohlin,
and Weinreb,
Inherited
Biophys.
294.
Hypotheses P. (1969) N. (1974)
Kitt, EngI.
Biochem. Chem.
(1974)
Pathol.
(1976)
New
(1971)
J. and
Gordon,
and
(1964)
J. Biol.
Silverstein,
of
McGraw-Hill,
Disease New
Res.
(Stanbury,
York
Commun.
18.
478A
J. Med.
J. Clin. T.,
H.A.
(1975)
21
Engl.
Am.
R.L.
22
Res.
2820)
1442-1444
295A
E. (1976)
Davidson.
M. and Soffer,
New
24.
PP. 730-759, 24,
Lieberman,
(No. Basis
Clin.
Weinreb,
Clin.
717
D. (1965)
14
(1976)
35,
TheMetabolic
eds.),
13
N.J.
Proc.
(1972)
250,
J. Exp.
66.
Am.
M.,
(1976)
J. Med. Pharmacol.
Clin.
Chem. Res.
22.
Pathol.
Ann. 270. 20,
Int.
Med.
139,
1637-1648
15,
61-4
391A
1228-1248
in press
Med.,
269-274
67626768
2, 75-78 Clin.
4.16-424
J. Clin.
submitted
221-225
J.B.,
Clinica Chimica Acta, 74 (1977) 21-25 0 Elsevier/North-Holland Biomedical Press
CCA 8138
ELEVATED
SERUM AND SPLEEN ANGIOTENSIN
ENZYME
AND SERUM LYSOZYME
EMANUEL
SILVERSTEIN
CONVERTING DISEASE
* and JOAN FRIEDLAND
Laboratory of Molecular Biology, Department Biochemistry, State University of New York, N.Y. 11203 (U.S.A.)
(Received
IN GAUCHER’S
of Medicine and Graduate Downstate Medical Center,
Program in Brooklyn,
June 4th, 1976)
Summary In adult chronic non-neuronopathic (Type 1) Gaucher’s disease significant (p < 0.001) elevations of angiotensin converting enzyme in serum (93.3 f 14.8 nmol/min/ml; number elevated, 8/11; normal control 32.2 + 1.30, n = 58) and spleen (5.62 f 0.35 nmol/min/mg protein, n = 2; control, 0.431 + 0.101, n = 4) and serum lysozyme (15.6 f 3.37 pg/ml; number elevated, 4/5) were observed. The KM for hippuryl-L-histidyl-L-leucine of Gaucher (1.31 mM) and normal (1.23 mM) serum angiotensin converting enzyme were similar. The increased angiotensin converting enzyme (ACE) in Gaucher’s disease may be related to the genetic defect resulting in increased ACE synthesis in Gaucher cells, or perhaps generally, while high lysozyme may reflect an increased body mass of reticuloendothelial cells. These enzyme elevations may be of use in suggesting the possible presence of Gaucher’s disease and perhaps in assessing the magnitude of pathologic involvement.
Introduction Angiotensin converting enzyme (ACE) and lysozyme are elevated and positively correlated in the serum [l-6] and granulomatous lymph nodes [3,6--81 in sarcoidosis, perhaps due to active synthesis by the sarcoid granulomas which contain epithelioid cells of the reticuloendothelial series. While ACE was not elevated in rat, mouse and rabbit macrophages, rat Freund’s adjuvant granulomas [9] and in human blood leukocytes and lymph nodes in a variety of diseases [ 81, we have found striking elevation of serum and spleen ACE and serum lysozyme in Gaucher’s disease which is characterized by massive accumulation of large characteristic reticuloendothelial cells (Gaucher’s cells) laden with glu* To
whom
correspondence
should
be addressed.
cocerebroside due to deficient glucocerebroside-/3-glucosidase [ 10,111. An abstract of this work [ 121, a brief paper on elevated ACE [ 131 and an abstract on elevated lysozyme [ 141 in Gauchcr’s disease have been reported. Materials and methods ACE was assayed fluorimetrically with the substrate hippuryl-L-histidyl-Lleucine [ 151. Lysozyme was determined turbidimetrically with the substrate Micrococcus lysodeihticus [16] and is given in terms of micrograms hen egg white lysozyme equivalents. Sera were obtained from blood samples which were allowed to clot for about 1 h at room temperature and were stored at -85°C until use. The preparation of tissue extracts for lysozyme assay is described elsewhere [ 161. Tissues for ACE assay were fractionated at 0--5°C from 5% homogenates in 50 mM potassium phosphate, pH 8.3, prepared by 40 seconds (total) homogenization in a tissue homogenizer. The 254 X g (10 min centrifugation) and 39 000 X g (1 h centrifugation) pellets were washed once by centrifugation and resuspended to the original homogenate volume by brief homogenization. The two pellets and the final supernatant were assayed for ACE. The diagnosis of Gaucher’s disease was made in all cases by the finding of Gaucher cells in tissue specimens, as well as by determination of diminished tissue P-glucosidase in most cases. Results and discussion The mean serum ACE in 11 patients with Gaucher’s disease was significantly elevated about 3 fold (Table I) as compared to normal subjects (p < 0.001). Two of the three subjects with serum ACE in the normal range were asymptomatic, while the third had severe disease and was the only subject on steroid therapy. Perhaps steroid therapy suppressed an elevated serum ACE level in the third patient, since steroid therapy usually suppresses elevated serum ACE levels in sarcoidosis [ 171. ACE in two Gaucher spleens was significantly elevated about 13 fold compared to control tissues (p < 0.001). Most of the ACE activity was particulate in both Gaucher and control spleens (Table II). Numerous Gaucher cells were present in the Gaucher spleens. No blood leukocyte ACE activity was detected in 2 patients with normal serum ACE. The Klvr for hippurylL-histidyl-L-leucine of serum ACE from a Gaucher’s disease patient with elevated serum ACE (1.31 mM) was similar to that of normal serum ACE (1.23 mM). Serum lysozyme was elevated in 4 of 5 patients studied (Table I). Lysozyme generally paralleled ACE and was normal in a patient with a low normal ACE. Lysozyme in two spleens from subjects with Gaucher’s disease was in the midrange of values for control spleens (Table III), which normally contain abundant lysozyme-rich reticuloendothelial cells. The serum ACE results in nine adult patients and spleen ACE values in one Gaucher patient and four controls which have recently been reported [ 131 are in general agreement with results here given, although the Gaucher spleen ACE was about half that found in our two cases, and the reported KM for hippuryl-
23 TABLE
I
SERUM
ANGIOTENSIN
Patient
* -
NO.
Age
Sex
1
42
F
2 3 4 5
12 29 50 45
n9
6
CONVERTfNG
ACE (nmol/min/ml)
ENZYME
AND LYSOZYME
Clinical description
Lysozyme
5.5
44.1 59.5 84.0 178
14.5
M M M
63
M
105
7 8 9 10
70 48 65 48
F F F M
31.0 145 138 97.6
10.8 29.3 13.0
11
18
F
107
20.0
32.2 t 1.30 (n = 58) [151 12.5-51.9 93.3 i 14.8
fi.4 (0 = 50) [I81 O-l 0 15.5 + 3.37
8111
5f6
* All patients
were white except
DISEASE
Qg/mU
26.2
Normal mean !z S.E.M Normal range Mean Gauchar’s disease Number Elevated
IN GAUCHER’S
Progressive hepatosplenomegaly. bone pain: on steroids AsymBtomatic: icteric: past splenectomy Asymptotic: minimal splenomegaly Asymptotic: marked throbocytopenia; splenomegaiy Moderate bone disease: chronic renal disease on dialysis: splenectomv Substantial bone disease, anemia. pulmonary emphysema, spfenectomy ASympt#matiC Severe, bone involvement; anemia Splenomegaly; benign monoclonal gammopathv Moderate splenomegaly, no Gaucher’s disease symptams: recent nephrotic syndrome Symptomntic. splenectomy
No. 5 who was black.
L-histidine-b-leucine was about 10.fold lower than we have found for human serum and 17-fold lower than the rabbit lung ACE value [ 19,201. The LO-fold lower control spleen lysozyme as compared to Gaucher spleen lysozyme which has been reported [ 141 contrasts with equal values in this study. Since we have observed [ 161 that extraction of the strongly basic molecule lysozyme from various tissues at neutral pH was strongly dependent on TABLE
II
ELEVATION SEASE
OF
Condition
Gaucher’s Gaucher’s
ANGIOTENSIN
Angiotensin
disease disease
Normal Normal Diabetes Tuberculosis
converting
ENZYME
enzyme
254X8 pellet
39 000 x &! pellet
8.09 3.47 0.52 0.32 0.88
Gaucher f. S.E.M @ < 0.0011 Mean control * S.E.M. Mean
CONVERTING
IN THE
SPLEEN
IN GAUCHEK’S
~~rnol~min~mg protein)
_I-
Supernatant
Total
23.9 30.0
2.26 0.55
5.27 5.97
3.06 2.12 1.17 4.00
0.20 0.02 0.06
0.68 0.32 0.22 0.50
_-
5.62 + 0.350 0.43 i 0.101 -1-
DI-
TABLE
III
SPLEEN ._
LYSOZYME
IN GAUCIIER’S
Condition
DISEASE
Lysozyme
Gaucher’s
disease
1
4.94
Gaucher’s
disease
2
5.17
Mean
Gaucher’s
disease
5.06
Normal
8.01
Diabetes
4.30
Bronchopneumonia
1.21
NOrmaI
6.46
Tuberculosis
5.51
Mean
5.10
control
AND
(I.rg/mg
CONTROLS
protein)
+ 0.115
i
1.14
ionic strength, 1 M NaCl was used for lysozyme extraction in the present study. We have found that about 2-3s of Gaucher spleen lysozyme extractable with 1 M NaCl is extractable in distilled water in a 5% homogenate in contrast to virtually no extraction of control spleen lysozyme, probably accounting for the reported elevated Gaucher spleen lysozyme [ 141. Increased serum lysozyme may result from synthesis by an increased body mass of reticuloendothelial cells [ 211. Increased ACE may be related to the genetic defect in Gauche& disease which may result in increased synthesis of ACE generally or perhaps specifically in Gaucher’s cells, perhaps analogous to the mechanism hypothesized for sarcoidosis [ 221. These increased serum enzymes, particularly ACE, may have diagnostic value in Gaucher’s disease and the level of increase may reflect the magnitude of disease and the severity of the defect. The relationship of the elevation of ACE, which is not a lysozomal enzyme, to that of various acid hydrolases [23] and transcobalamin II [ 241 is not clear. Acknowledgments We are indebted to Dr. Neal Weinreb, Dr. Arthur Sawitsky, Dr. Irving Swiller, Dr. Stanley Lee, Mrs. Phyllis Schwartz and Mrs. Fran Berkwits for Gaucher serum samples, Dr. Arthur Sawitsky for Gaucher spleen samples, and Dr. Yale Rosen for histologic sections. We thank Charlotte Setton for able technical assistance. References Lieberman,
J.(1975)
Silverstein,
E.,
Silverstein, and
I?.,
Other
Friedland, Pascual,
Am.
Friedland,
J. Med. J..
Friedland,
J.,
Granulomatous Gee.
J.B.L.
Lyons.
Disorders.
J. and Silverstein, R.S.,
59,
Lyons,
E. (1976) and Finch.
Silverstein.
E..
Friedland,
J. and
Silverstein,
E.,
Friedland.
J., Lyons,
Silverstein,
E..
2137-2141
Friedland.
J..
365-372 H. and Kitt, H.A.
and
Ann.
N.Y. (1973)
H. and H.A.
T.
(1975)
Acad.
Biochem. S.C.
Ackerman,
Lyons.
M.
Gourin,
and
278.
Med.
15.
178-185
New.
Eng. Clin.
A.
Gourin,
Res.
(1976)
Sci.,
(1975)
Gourin,
Clin. A.
(1975) A.
352A Int.
Conf.
on
Sarcoidosis
498-513
J. Med. Res.
23.
Seventh
23,
Clin. (1976)
289,
1074-1076
600A Res. Proc.
23.
352A
Natl.
Acad.
Sci.
U.S.A.
73,
25
9 10
Silverstein,
E. and
Fredrickson.
D.S.
Wyngaarden,
J.B.
11
Brady,
12
Silverstein,
R.O.,
Friedland, and and
Kanfer,
J. (1976)
Sloan,
H.R.
Fredrickson. J.N.
and
E. and
Friedland,
J. and
Beutler.
Fed.
D.S.,
Shapiro. J. (1976)
E. (1976)
Biochem.
15
Friedland,
16
Silverstein,
E.,
Friedland.
J. and Ackerman,
17
Silverstein.
E.,
Friedland.
J., Lyons.
18
Prockop.
19
Cushman,
20
Das,
Res.
J. and Silverstein,
D.J.
and
D.W.
W.D.
and Cheung,
H.S.
S..
Z.A.
23
Ockerman,
P.A.
24
Gilbert,
H.S.
Todd,
Cohn,
E. (1976)
Med.
and Kohlin,
and Weinreb,
Inherited
Biophys.
294.
Hypotheses P. (1969) N. (1974)
Kitt, EngI.
Biochem. Chem.
(1974)
Pathol.
(1976)
New
(1971)
J. and
Gordon,
and
(1964)
J. Biol.
Silverstein,
of
McGraw-Hill,
Disease New
Res.
(Stanbury,
York
Commun.
18.
478A
J. Med.
J. Clin. T.,
H.A.
(1975)
21
Engl.
Am.
R.L.
22
Res.
2820)
1442-1444
295A
E. (1976)
Davidson.
M. and Soffer,
New
24.
PP. 730-759, 24,
Lieberman,
(No. Basis
Clin.
Weinreb,
Clin.
717
D. (1965)
14
(1976)
35,
TheMetabolic
eds.),
13
N.J.
Proc.
(1972)
250,
J. Exp.
66.
Am.
M.,
(1976)
J. Med. Pharmacol.
Clin.
Chem. Res.
22.
Pathol.
Ann. 270. 20,
Int.
Med.
139,
1637-1648
15,
61-4
391A
1228-1248
in press
Med.,
269-274
67626768
2, 75-78 Clin.
4.16-424
J. Clin.
submitted
221-225
J.B.,