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The absence of γ-glutamyltransferase in erythrocyte membranes
Clinica Chimica Actu, 168 (1987) 347-349 Elsevier
347
CCA 03934
Letter to the editor
The absence of y-glutamyltransferase in erythrocyte membranes Dear Editor,
Daniel et al [l] reported that the activity of erythrocyte y-glutamyltransferase (y-glutamyl transpeptidase) in patients with hepatobiliary disease was elevated five-fold over healthy adult values. Ten years ago, Srivastava et al [2] reported that no activity of y-glutamyl transpeptidase could be detected in either human or rabbit erythrocytes. Subsequently, Board and Smith [3] and Marstein and Perry [4] confirmed this finding. Both Srivastava et al [2] and Board and Smith [3] concluded that the activity present in unfiltered samples can be attributed to leukocyte contamination. According to Daniel et al [l], the y-glutamyl transpeptidase activity in control erythrocytes averaged 0.197 nmol substrate/min/mg ghost protein at 37°C; the highest value observed was 6 mnol/min/mg protein in patients with liver carcinoma. Board and Smith [3] determined the enzyme activity by a radioisotopic method, using the same substrate, y-glutamyl-p-nitroanilide, which Daniel et al used, and reported that the unfiltered erythrocytes contained 2.15 nmol/min/mg protein but that red cells filtered to remove leukocytes contained only 0.05 nmoles/min/mg protein. We suggest that Daniel et al [l] were misled by the existence of white blood cell contamination in their red cell preparations because they failed to filter the whole blood to remove leukocytes before their assay. Washing of erythrocytes only removes approximately 60-90% of the leukocytes [2,5]. Although we do not know if it was the number of leukocytes or their enzyme activity that is increased in the diseases studied by Daniel et al [l], they need to demonstrate that, in fact, they measure human erythrocyte y-glutamyl transpeptidase.
References 1 Daniel DS, Ramakrishna BS, Balasubramanian KA. Human erythrocyte y-ghnamyltransferase in liver diseases. Chn Chim Acta 1987;162:319-327. 2 Srivastava SK, Awasthi YC, Miller SP, Yoshida A, Beutler E. Studies on y-glutamyl transpeptidase in human and rabbit erythrocytes. Blood 1976;47:645-650. 3 Board PG, Smith JE. Erythrocyte y-glutamyl transpeptidase. Blood 1977;49:667-668. 0009-8981/87/$03.50
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
348 4 Marstein S, Perry TL. Studies of amino acid content and transport in glutathione-deficient erythrocytes from a patient with pyroglutamic acidemia (5-oxoprolinemia). Clin Chim Acta 1981;109:13-20. 5 Beutler E, West C, Blume K-G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med 1976:88:328-333.
Takashige Suzuki, George L. Dale and Ernest Beutler Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037, U.S.A.
(Received
Authors’
23 March
1987)
reply
Dear Editor, We thank Suzuki et al for pointing out the possibility of leukocyte contamination contributing to erythrocyte y-glutamyltransferase (GGT) measured. We found that GGT activity in erythrocyte ghosts prepared by method of Fairbanks et al [l] from blood of patients with liver disease usually, but not always, was increased [2]. We wanted to place this key observation on record since erythrocyte GGT activity in liver disease has not been studied so far. The pathophysiologic mechanism responsible for this observation needs to be explored. The leukocyte poor erythrocytes used for preparation of ghost had about 100 leukocytes/cumm packed erythrocytes. This could be achieved by sacrificing 20% erythrocytes while removing the buffy-coat. Filtration through cotton-wool did not help removal of this minimal contamination and there was no significant difference in the activity measured (Table I). The activity in unfiltered erythrocytes reported by Board and Smith [3] was ten times that reported for controls by us which is close to that of filtered erythrocytes. GGT activity in one of the controls was 2.8 x 10e4 U/mg ghost protein when leukocyte count was 5000 cells/cumm blood and
TABLE
Patient
I
y-Glutamyltransferase Removal
A B e D
3.14 6.79 8.82 7.22
of buffy-coat
( x 10m4 U/mg
ghost protein) Filtration 5.00 5.58 8.82 10.23
in erythrocytes
through
cotton
wool
after:
349
3.9 x 10e4 U/mg at a different occasion when it was 69000 leukocytes/cumm blood due to acute responsive eosinophilia. However, we cannot exclude the possibility of increased leukocyte GGT activity in liver disease even though we have taken care to minimise the number of leukocytes contaminating our erythrocyte ghost preparations. References 1 Fairbanks G, Steck TL, Wallach DFH. Electrophoretic analysis of major polypeptides erythrocyte membrane. Biochemistry 1971;10:2606-2617. 2 Daniel DS, Ramakrishna BS, Balasubramanian KA. Human erythrocyte y-ghrtamyltransferase diseases. Clin Chim Acta 1987;162:319-327. 3 Board PG, Smith JE. Erythrocyte y-glutamyltranspeptidase. Blood 1977;49:667-668.
of human in liver
D. Sundarsingh Daniel, B.S. Ramakrishna and K.A. Balasubramanian Wellcome Research Unit, Christian Medical College, Yellore 632 004, India
(Received
5 May 1987)
347
CCA 03934
Letter to the editor
The absence of y-glutamyltransferase in erythrocyte membranes Dear Editor,
Daniel et al [l] reported that the activity of erythrocyte y-glutamyltransferase (y-glutamyl transpeptidase) in patients with hepatobiliary disease was elevated five-fold over healthy adult values. Ten years ago, Srivastava et al [2] reported that no activity of y-glutamyl transpeptidase could be detected in either human or rabbit erythrocytes. Subsequently, Board and Smith [3] and Marstein and Perry [4] confirmed this finding. Both Srivastava et al [2] and Board and Smith [3] concluded that the activity present in unfiltered samples can be attributed to leukocyte contamination. According to Daniel et al [l], the y-glutamyl transpeptidase activity in control erythrocytes averaged 0.197 nmol substrate/min/mg ghost protein at 37°C; the highest value observed was 6 mnol/min/mg protein in patients with liver carcinoma. Board and Smith [3] determined the enzyme activity by a radioisotopic method, using the same substrate, y-glutamyl-p-nitroanilide, which Daniel et al used, and reported that the unfiltered erythrocytes contained 2.15 nmol/min/mg protein but that red cells filtered to remove leukocytes contained only 0.05 nmoles/min/mg protein. We suggest that Daniel et al [l] were misled by the existence of white blood cell contamination in their red cell preparations because they failed to filter the whole blood to remove leukocytes before their assay. Washing of erythrocytes only removes approximately 60-90% of the leukocytes [2,5]. Although we do not know if it was the number of leukocytes or their enzyme activity that is increased in the diseases studied by Daniel et al [l], they need to demonstrate that, in fact, they measure human erythrocyte y-glutamyl transpeptidase.
References 1 Daniel DS, Ramakrishna BS, Balasubramanian KA. Human erythrocyte y-ghnamyltransferase in liver diseases. Chn Chim Acta 1987;162:319-327. 2 Srivastava SK, Awasthi YC, Miller SP, Yoshida A, Beutler E. Studies on y-glutamyl transpeptidase in human and rabbit erythrocytes. Blood 1976;47:645-650. 3 Board PG, Smith JE. Erythrocyte y-glutamyl transpeptidase. Blood 1977;49:667-668. 0009-8981/87/$03.50
0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
348 4 Marstein S, Perry TL. Studies of amino acid content and transport in glutathione-deficient erythrocytes from a patient with pyroglutamic acidemia (5-oxoprolinemia). Clin Chim Acta 1981;109:13-20. 5 Beutler E, West C, Blume K-G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med 1976:88:328-333.
Takashige Suzuki, George L. Dale and Ernest Beutler Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037, U.S.A.
(Received
Authors’
23 March
1987)
reply
Dear Editor, We thank Suzuki et al for pointing out the possibility of leukocyte contamination contributing to erythrocyte y-glutamyltransferase (GGT) measured. We found that GGT activity in erythrocyte ghosts prepared by method of Fairbanks et al [l] from blood of patients with liver disease usually, but not always, was increased [2]. We wanted to place this key observation on record since erythrocyte GGT activity in liver disease has not been studied so far. The pathophysiologic mechanism responsible for this observation needs to be explored. The leukocyte poor erythrocytes used for preparation of ghost had about 100 leukocytes/cumm packed erythrocytes. This could be achieved by sacrificing 20% erythrocytes while removing the buffy-coat. Filtration through cotton-wool did not help removal of this minimal contamination and there was no significant difference in the activity measured (Table I). The activity in unfiltered erythrocytes reported by Board and Smith [3] was ten times that reported for controls by us which is close to that of filtered erythrocytes. GGT activity in one of the controls was 2.8 x 10e4 U/mg ghost protein when leukocyte count was 5000 cells/cumm blood and
TABLE
Patient
I
y-Glutamyltransferase Removal
A B e D
3.14 6.79 8.82 7.22
of buffy-coat
( x 10m4 U/mg
ghost protein) Filtration 5.00 5.58 8.82 10.23
in erythrocytes
through
cotton
wool
after:
349
3.9 x 10e4 U/mg at a different occasion when it was 69000 leukocytes/cumm blood due to acute responsive eosinophilia. However, we cannot exclude the possibility of increased leukocyte GGT activity in liver disease even though we have taken care to minimise the number of leukocytes contaminating our erythrocyte ghost preparations. References 1 Fairbanks G, Steck TL, Wallach DFH. Electrophoretic analysis of major polypeptides erythrocyte membrane. Biochemistry 1971;10:2606-2617. 2 Daniel DS, Ramakrishna BS, Balasubramanian KA. Human erythrocyte y-ghrtamyltransferase diseases. Clin Chim Acta 1987;162:319-327. 3 Board PG, Smith JE. Erythrocyte y-glutamyltranspeptidase. Blood 1977;49:667-668.
of human in liver
D. Sundarsingh Daniel, B.S. Ramakrishna and K.A. Balasubramanian Wellcome Research Unit, Christian Medical College, Yellore 632 004, India
(Received
5 May 1987)