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LEUCOCYTE G.-6-P.D. DEFICIENCY
110 TABLE I-GLYCOLYTIC AND HEXOSE-MONOPHOSPHATE-SHUNT ACTIVITY OF LEUCOCYTES FROM PATIENT F.E. AND CONTROLS*
specimens and propagated in the usual manner.10 They were stained with toluidine-blue-0 by the technique of Danes and Beam.7 At the fourth transfer-the earliest assessment - definite metachromasia in a large number of cells was observed. At the fifth transfer, 70% of the cells (1000 cells counted) were metachromatic. Between 5 to 10% of control cells in parallel cultures showed metachromasia. While we cannot claim unambiguously that " Lafora bodies " are present in cultured fibroblasts, certain unusual features of these cells are noteworthy. There were many irregularly shaped metachromatic inclusion bodies. Nuclei were frequently distorted, often kidney-shaped, and were displaced toward the periphery of the cell. The kidneyshaped nuclei appeared to be deformed around an illdefined mass of granular material occupying the centre of the cell which stained bluer and more heavily than the remaining cytoplasmic material and nuclei. We have never observed similar structures in other metachromatic cultures-e.g., mucopolysaccharidoses. There were no aberrations of chromosome number. While it is a truism that metachromasia per se is of questionable value as a diagnostic tool," the unusual morphological features observed in the present culture, coupled with the known chemistry of the disease, provides a logical rationale for the metachromasia. Thus, fibroblasts from patients with Lafora’s disease can be expected to serve as a pathognomonic model for the investigation of the basic biochemical abnormality of the disease. ARVAN L. FLUHARTY MYNA T. PORTER Research Department, GRACE A. HIRSH Pacific State Hospital, EMILIO PEVIDA Box 100, HAYATO KIHARA. Pomona, California 91766.
LEUCOCYTE G.-6-P.D. DEFICIENCY SIR,-We have followed with interest the postulate of Dr. Schlegel and Dr. Bellanti 12 stating that the deficiency of
leucocyte glucose-6-phosphate dehydrogenase (G.-6-P.D.) for increased susceptibility of males to infection. Dr. G. E. Rodey and his colleagues 13 have recently described a patient in whom the level of leucocyte G.-6-P.D. was depressed to 25% of normal. These investigators demonstrated that the metabolic activity of these cells did not differ significantly from control cells in either the resting or the phagocytising state. Further, the ability of the G.-6-P.D.deficient cells to kill staphylococci was normal, although the rate of phagocytosis was somewhat impaired. These data were presented as evidence against a critical role for leucocyte G.-6-P.D. in phagocytic and bactericidal activity of accounts
/eucocytes. We have lately studied a patient (Caucasian, female )14 whose clinical picture was characterised by hxmolytic anxmia, a leuka:moid reaction, and severe recurrent infections. Leucocyte ingestion of bacteria was normal by plate dilution techniques. Latex and ’Zymozan ’ particles were
phagocytised normally. glucose utilisation by the method
Measurement of
Holmes
et
*
Values are expressed as c.p.m. of "C released by 5 x 106 leucocytes. The control data, expressed as the mean ± standard deviation, represents an average of 20 determinations; the patient’s data, expressed simply as the mean, represents an average of 4 separate determinations.
the patient’s cells, but a total lack of hexose-monophosphateshunt activity in either the resting or phagocytising state (table I). It was further observed that the addition of methylene-blue failed to stimulate the hexose-monophosphate shunt in the patient’s leucocytes. The activities of the first two shunt enzymes, G-6-P.D. and 6-phosphogluconic acid dehydrogenase (6-P.G.A.D.), were determined in sonicates of control and the patient’s leucocytes by conventional methods.16,17 Table 11 demonstrates that the patient’s polymorphonuclear leucocytes completely lacked G-6-P.D. activity, while possessing normal 6-P.G.A.D. activity. A number of other enzymes (including pyruvic kinase, N.A.D.H. - oxidase, and N.A.D.P.G. oxidase) showed TABLE
FROM PATIENT
*
Values expressed as the and 6 patient samples.
mean
T., Fluharty, A. L., Kihara, H. Proc. natn. Acad. Sci. 1969, 62, 887. 11. Taysi, K., Kistenmacher, M. L., Punnett, H. H., Mellman, W. J. New Engl. J. Med. 1969, 281, 1108. 12. Schlegel, R. J., Bellanti, J. A. Lancet, 1969, ii, 826. 13. Rodey, G. E., Jacob, H. S., Holmes, B., McArthur, J. R., Good, R. A. ibid. 1970, i, 355. 14. Cooper, M. R., DeChatelet, L. R., McCall, C. E., LaVia, M. F., Spurr, C. L., Baehner, R. L. Paper read at 62nd Annual Meeting of the American Society for Clinical Investigation, Atlantic City, New Jersey, May 3, 1970; p. 21a (abstract). 15. Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A. Lancet,
ACTIVITIES
OF
LEUCOCYTES
F.E.*
::I:: standard deviation for 12 control
normal activity. The production of hydrogen peroxide (measured by formate-14C utilisation 18) was less than 25% of normal. Bactericidal activity of F.E.’s leucocytes was measured by standard plate-dilution techniques.19,20 The cells were found to have defective capacity to kill Staphylococcus aureus, Escherichia coli, and Serratia marcescens (bacteria which produce little or no hydrogen peroxide), although
Streptococcus faecalis (an H202-producing bacterium)
was
killed normally. These data strongly imply that the integrity of the hexosemonophosphate shunt is essential for the normal bactericidal activity of leucocytes; in this sense, G-6-P.D. does play a critical role in polymorphonuclear-leucocyte bacterial killing. The data of Dr. Rodey and his colleagues seem to indicate that the normal cell contains a considerable excess of this enzyme, and that a reduction to considerably less than 25% of the normal level is required before the amount of the enzyme becomes a limiting factor.
of
al.15 demonstrated normal sivcolvtic activitv in
Gray School of Medicine, Winston-Salem, North Carolina, and Children’s Hospital
Bowman
10. Porter, M.
1966, i, 1225.
II-G.-6-P.D. AND 6-P.G.A.D.
Medical Center, Boston, Massachusetts. 16.
17. 18. 19. 20.
M. ROBERT COOPER LAWRENCE R. DECHATELET CHARLES E. MCCALL MARIANO F. LAVIA CHARLES L. SPURR ROBERT L. BAEHNER.
L. in Methods in Enzymology (edited by S. P. Colowick and N. O. Kaplan); vol. I, p. 323. New York, 1955. Horecker, B. L., Smyrniotis, P. Z. ibid. p. 326. Iyer, G. Y. N., Islam, D. M. E., Quastel, J. A. Nature, Land. 1961, 192, 535. Maaloe, O. On the Relation between Alexin and Opsonin; p. 186. Copenhagen, 1946. Cohn, Z. A., Morse, S. I. J. exp. Med. 1959, 110, 419.
Kornberg, A. Horecker, B.
specimens and propagated in the usual manner.10 They were stained with toluidine-blue-0 by the technique of Danes and Beam.7 At the fourth transfer-the earliest assessment - definite metachromasia in a large number of cells was observed. At the fifth transfer, 70% of the cells (1000 cells counted) were metachromatic. Between 5 to 10% of control cells in parallel cultures showed metachromasia. While we cannot claim unambiguously that " Lafora bodies " are present in cultured fibroblasts, certain unusual features of these cells are noteworthy. There were many irregularly shaped metachromatic inclusion bodies. Nuclei were frequently distorted, often kidney-shaped, and were displaced toward the periphery of the cell. The kidneyshaped nuclei appeared to be deformed around an illdefined mass of granular material occupying the centre of the cell which stained bluer and more heavily than the remaining cytoplasmic material and nuclei. We have never observed similar structures in other metachromatic cultures-e.g., mucopolysaccharidoses. There were no aberrations of chromosome number. While it is a truism that metachromasia per se is of questionable value as a diagnostic tool," the unusual morphological features observed in the present culture, coupled with the known chemistry of the disease, provides a logical rationale for the metachromasia. Thus, fibroblasts from patients with Lafora’s disease can be expected to serve as a pathognomonic model for the investigation of the basic biochemical abnormality of the disease. ARVAN L. FLUHARTY MYNA T. PORTER Research Department, GRACE A. HIRSH Pacific State Hospital, EMILIO PEVIDA Box 100, HAYATO KIHARA. Pomona, California 91766.
LEUCOCYTE G.-6-P.D. DEFICIENCY SIR,-We have followed with interest the postulate of Dr. Schlegel and Dr. Bellanti 12 stating that the deficiency of
leucocyte glucose-6-phosphate dehydrogenase (G.-6-P.D.) for increased susceptibility of males to infection. Dr. G. E. Rodey and his colleagues 13 have recently described a patient in whom the level of leucocyte G.-6-P.D. was depressed to 25% of normal. These investigators demonstrated that the metabolic activity of these cells did not differ significantly from control cells in either the resting or the phagocytising state. Further, the ability of the G.-6-P.D.deficient cells to kill staphylococci was normal, although the rate of phagocytosis was somewhat impaired. These data were presented as evidence against a critical role for leucocyte G.-6-P.D. in phagocytic and bactericidal activity of accounts
/eucocytes. We have lately studied a patient (Caucasian, female )14 whose clinical picture was characterised by hxmolytic anxmia, a leuka:moid reaction, and severe recurrent infections. Leucocyte ingestion of bacteria was normal by plate dilution techniques. Latex and ’Zymozan ’ particles were
phagocytised normally. glucose utilisation by the method
Measurement of
Holmes
et
*
Values are expressed as c.p.m. of "C released by 5 x 106 leucocytes. The control data, expressed as the mean ± standard deviation, represents an average of 20 determinations; the patient’s data, expressed simply as the mean, represents an average of 4 separate determinations.
the patient’s cells, but a total lack of hexose-monophosphateshunt activity in either the resting or phagocytising state (table I). It was further observed that the addition of methylene-blue failed to stimulate the hexose-monophosphate shunt in the patient’s leucocytes. The activities of the first two shunt enzymes, G-6-P.D. and 6-phosphogluconic acid dehydrogenase (6-P.G.A.D.), were determined in sonicates of control and the patient’s leucocytes by conventional methods.16,17 Table 11 demonstrates that the patient’s polymorphonuclear leucocytes completely lacked G-6-P.D. activity, while possessing normal 6-P.G.A.D. activity. A number of other enzymes (including pyruvic kinase, N.A.D.H. - oxidase, and N.A.D.P.G. oxidase) showed TABLE
FROM PATIENT
*
Values expressed as the and 6 patient samples.
mean
T., Fluharty, A. L., Kihara, H. Proc. natn. Acad. Sci. 1969, 62, 887. 11. Taysi, K., Kistenmacher, M. L., Punnett, H. H., Mellman, W. J. New Engl. J. Med. 1969, 281, 1108. 12. Schlegel, R. J., Bellanti, J. A. Lancet, 1969, ii, 826. 13. Rodey, G. E., Jacob, H. S., Holmes, B., McArthur, J. R., Good, R. A. ibid. 1970, i, 355. 14. Cooper, M. R., DeChatelet, L. R., McCall, C. E., LaVia, M. F., Spurr, C. L., Baehner, R. L. Paper read at 62nd Annual Meeting of the American Society for Clinical Investigation, Atlantic City, New Jersey, May 3, 1970; p. 21a (abstract). 15. Holmes, B., Quie, P. G., Windhorst, D. B., Good, R. A. Lancet,
ACTIVITIES
OF
LEUCOCYTES
F.E.*
::I:: standard deviation for 12 control
normal activity. The production of hydrogen peroxide (measured by formate-14C utilisation 18) was less than 25% of normal. Bactericidal activity of F.E.’s leucocytes was measured by standard plate-dilution techniques.19,20 The cells were found to have defective capacity to kill Staphylococcus aureus, Escherichia coli, and Serratia marcescens (bacteria which produce little or no hydrogen peroxide), although
Streptococcus faecalis (an H202-producing bacterium)
was
killed normally. These data strongly imply that the integrity of the hexosemonophosphate shunt is essential for the normal bactericidal activity of leucocytes; in this sense, G-6-P.D. does play a critical role in polymorphonuclear-leucocyte bacterial killing. The data of Dr. Rodey and his colleagues seem to indicate that the normal cell contains a considerable excess of this enzyme, and that a reduction to considerably less than 25% of the normal level is required before the amount of the enzyme becomes a limiting factor.
of
al.15 demonstrated normal sivcolvtic activitv in
Gray School of Medicine, Winston-Salem, North Carolina, and Children’s Hospital
Bowman
10. Porter, M.
1966, i, 1225.
II-G.-6-P.D. AND 6-P.G.A.D.
Medical Center, Boston, Massachusetts. 16.
17. 18. 19. 20.
M. ROBERT COOPER LAWRENCE R. DECHATELET CHARLES E. MCCALL MARIANO F. LAVIA CHARLES L. SPURR ROBERT L. BAEHNER.
L. in Methods in Enzymology (edited by S. P. Colowick and N. O. Kaplan); vol. I, p. 323. New York, 1955. Horecker, B. L., Smyrniotis, P. Z. ibid. p. 326. Iyer, G. Y. N., Islam, D. M. E., Quastel, J. A. Nature, Land. 1961, 192, 535. Maaloe, O. On the Relation between Alexin and Opsonin; p. 186. Copenhagen, 1946. Cohn, Z. A., Morse, S. I. J. exp. Med. 1959, 110, 419.
Kornberg, A. Horecker, B.