- Email: [email protected]
COPPER DEFICIENCY
1488 COPPER DEFICIENCY
SIR,-Your April 18 editorial (p 900) mentions bradycardia as a feature of copper deficiency in the rat. Indeed this was an effect of copper deficiency, as reported by Prohaska and Heller,’ but is misleading in the context of a review of copper deficiency in man. Supraventricular tachycardia not bradycardia has been reported in human beings fed a diet containing 0-8 mg copper per day under metabolically controlled conditions (recommended copper intake is 2-3 mg per
day2,3).
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, STEPHEN CUNNANE Toronto, Ontario M5S 1A8, Canada 1. Prohaska JR, Heller LJ. Mechanical properties of the copper deficient rat heart. J Nutr 1982, 112: 2142-50. 2. Reiser S, Smith JC Jr, Mertz W, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. Am J Clin Nutr 1985; 42: 242-51.
3.
Klevay LM, Inman L, Johnson LK, et al. Increased cholesterol in plasma in a young man during experimental copper depletion. Metabolism 1984; 33: 1112-18.
ANTI-NEUTROPHIL-CYTOPLASM ANTIBODIES IN WEGENER’S GRANULOMATOSIS ARE NOT DIRECTED AGAINST ALKALINE PHOSPHATASE
SIR,-Dr Lockwood and colleagues (March 28, p 716) propose an
association between alkaline
phosphatase and
an
autoantigen
recognised by circulating anti-neutrophil antibodies in systemic vasculitis. They identify these antibodies as the anti-cytoplasmic or anti-neutrophil-cytoplasm (ANCA) antibodies described by van der Woude et aP and Rasmussen and Wiik2 as being specific for Wegener’s granulomatosis (WG). We are working, with several other groups, on the identification of the ANCA-related antigen but have not found any association between ANCA-related antigen and alkaline phosphatase in subcellular fractions of human neutrophils obtained by ’Percoll’ gradient centrifugation33 (figure). ANCA-related antigen is identified by ELISA. One reason for this discrepancy may be methodological. In contrast to our method for preparation of cytoplasmic constituents (nitrogen bomb cavitation in relaxation buffer with protease inhibitors), the neutrophil acid extract investigated by Lockwood et al is highly degraded. Indeed, most of their extract has a molecular weight below 6 kD (figs 2 and 3 in Lockwood’s paper). Slight deviation from the correct handling of our preparations also yields degraded material. Normal serum may react in ELISA with several ’Sephadex G-100’ fractions from degraded preparations. This applies especially to the 100 kD peak identified by Lockwood et al as an ANCA-specific peak. The diffuse binding of WG serum to all fractions (as in Lockwood’s fig 3) is familiar to us when we use degraded material. The very high concentration of serum (1 :4) used in their solid phase radioimmunoassay may also cause non-specific
binding. Besides the difficulty with the specificity of binding of WG serum (discussed above) there are other problems. One argument for an
association between alkaline phosophatase and the ANCA-related antigen is that the monoclonal antibody W8, raised against the 6-2 kD fraction of the neutrophil acid extract, binds to the same fractions as WG serum. However, the specificity of this binding has not been demonstrated. Moreover, the inhibition (or displacement) experiments are difficult to interpret because the meaning of "equivalent concentrations" of bovine serum albumin and alkaline
phosphatase is not clear. A simpler approach is to
use the original method for detecting ANCA.2 On indirect immunofluorescence with rabbit anti-human
alkaline
even, phosphatase (AP 268, Dakopatts, Copenhagen) homogeneous, "cytoplasmic" staining of the entire cell membrane (which does not cover the nucleus, when the smear technique described2 is used) is seen. This contrasts with the uneven granular staining of the cytoplasm seen with ANCA-containing serum. Moreover, preincubation of the smears with AP 268 or ANCAcontaining serum does not influence the binding of the other. Alkaline phosphatase is located at the cell membrane4 (the an
ELISA-identified binding of IgG from WG and control serum diluted 1 :80 to percoll gradient fractions of human neutrophil cytoplasm characterised by biochemical markers including alkaline phosphatase.
fractions
containing alkaline phosphatase are those containing the membranes3) but ANCA is directed against a cellular compartment with a density between that of the primary and secondary granules of the neutrophils (repeated and reproduced with nine different preparations and 14 different sera), as seen in the accompanying figure. Whether this ANCA-related antigen is located on a separate type of granule is not yet established. Since ANCA is specific for WG (provided that microscopic polyarteritiss is classified as a subgroup of WG) identification of the ANCA-related antigen may prove to be an important step in the elucidation of the pathogenesis of WG and/or microscopic polyarteritis. Our observations do not exclude the possibility that ANCA can react or be associated with alkaline phosphatase but they strongly suggest that ANCA-related antigen is not the enzyme itself. N. R. is supported by Weimann’s Legat and the Danish Arthritis Foundation (grant 233-490). N. B. is supported by the Danish Medical Research Council (grant 12-6139), the Danish Cancer Society (grant 86-132), and the Danish Arthritis Foundation (grant 233-627).
Laboratory of Autoimmune Serology, State Serum Institute, DK-2300 Copenhagen S Denmark; and Department of Medicine and Haematology C, Gentofte University Hospital, Hellerup
NIELS RASMUSSEN NIELS BORREGAARD ALLAN WIIK
der Woude FJ, Rasmussen N, Lobatto S, et al. Autoantibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity m Wegener’s granulomatosis. Lancet 1985; i: 425-29. 2. Rasmussen N, Wiik A. Autoimmunity in Wegener’s granulomatosis. In: Veldman JE, McCabe BF, Huizing EH, Mygind N, eds. Immunobiology, autoimmunity, transplantation, in otorhinolaryngology. Amsterdam. Kugler, 1985: 207-12. 3. Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: Translocation during activation. J Cell Biol 1983; 97: 52-61. 4. Borgers M, Thone F, de Cree J, de Cock W. Alkaline phosphatase activity in human polymorphonuclear leukocytes. Histochem J 1978; 10: 31-43. 5. Savage COS, Winearls CG, Evans DJ, Rees AJ, Lockwood CM. Microscopic polyarteritis. Presentation, pathology and prognosis. Quart J Med 1985; 56: 467-84. 1.
van
SIR,-Antibodies against cytoplasmic antigens of neutrophil granulocytes and monocytes (ACPA) are of value both for screening and for monitoring disease activity in Wegener’s granulomatosis (WG).l,2 The strong association between titre and disease activity suggests that these autoantibodies may be involved in the pathogenesis of WG. On the other hand, the fact that the target antigen is intracellular, and thus not accessible to circulating antibodies, casts doubt on the immunopathogenic relevance of ACPA in vivo. Dr Lockwood and colleagues claim that the target antigen is associated with alkaline phosphatase, which is partly located in the cytoplasmic membrane of neutrophils and is thus accessible to ACPA. However, our findings argue against the conclusion that the target antigen is alkaline phosphatase.
SIR,-Your April 18 editorial (p 900) mentions bradycardia as a feature of copper deficiency in the rat. Indeed this was an effect of copper deficiency, as reported by Prohaska and Heller,’ but is misleading in the context of a review of copper deficiency in man. Supraventricular tachycardia not bradycardia has been reported in human beings fed a diet containing 0-8 mg copper per day under metabolically controlled conditions (recommended copper intake is 2-3 mg per
day2,3).
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, STEPHEN CUNNANE Toronto, Ontario M5S 1A8, Canada 1. Prohaska JR, Heller LJ. Mechanical properties of the copper deficient rat heart. J Nutr 1982, 112: 2142-50. 2. Reiser S, Smith JC Jr, Mertz W, et al. Indices of copper status in humans consuming a typical American diet containing either fructose or starch. Am J Clin Nutr 1985; 42: 242-51.
3.
Klevay LM, Inman L, Johnson LK, et al. Increased cholesterol in plasma in a young man during experimental copper depletion. Metabolism 1984; 33: 1112-18.
ANTI-NEUTROPHIL-CYTOPLASM ANTIBODIES IN WEGENER’S GRANULOMATOSIS ARE NOT DIRECTED AGAINST ALKALINE PHOSPHATASE
SIR,-Dr Lockwood and colleagues (March 28, p 716) propose an
association between alkaline
phosphatase and
an
autoantigen
recognised by circulating anti-neutrophil antibodies in systemic vasculitis. They identify these antibodies as the anti-cytoplasmic or anti-neutrophil-cytoplasm (ANCA) antibodies described by van der Woude et aP and Rasmussen and Wiik2 as being specific for Wegener’s granulomatosis (WG). We are working, with several other groups, on the identification of the ANCA-related antigen but have not found any association between ANCA-related antigen and alkaline phosphatase in subcellular fractions of human neutrophils obtained by ’Percoll’ gradient centrifugation33 (figure). ANCA-related antigen is identified by ELISA. One reason for this discrepancy may be methodological. In contrast to our method for preparation of cytoplasmic constituents (nitrogen bomb cavitation in relaxation buffer with protease inhibitors), the neutrophil acid extract investigated by Lockwood et al is highly degraded. Indeed, most of their extract has a molecular weight below 6 kD (figs 2 and 3 in Lockwood’s paper). Slight deviation from the correct handling of our preparations also yields degraded material. Normal serum may react in ELISA with several ’Sephadex G-100’ fractions from degraded preparations. This applies especially to the 100 kD peak identified by Lockwood et al as an ANCA-specific peak. The diffuse binding of WG serum to all fractions (as in Lockwood’s fig 3) is familiar to us when we use degraded material. The very high concentration of serum (1 :4) used in their solid phase radioimmunoassay may also cause non-specific
binding. Besides the difficulty with the specificity of binding of WG serum (discussed above) there are other problems. One argument for an
association between alkaline phosophatase and the ANCA-related antigen is that the monoclonal antibody W8, raised against the 6-2 kD fraction of the neutrophil acid extract, binds to the same fractions as WG serum. However, the specificity of this binding has not been demonstrated. Moreover, the inhibition (or displacement) experiments are difficult to interpret because the meaning of "equivalent concentrations" of bovine serum albumin and alkaline
phosphatase is not clear. A simpler approach is to
use the original method for detecting ANCA.2 On indirect immunofluorescence with rabbit anti-human
alkaline
even, phosphatase (AP 268, Dakopatts, Copenhagen) homogeneous, "cytoplasmic" staining of the entire cell membrane (which does not cover the nucleus, when the smear technique described2 is used) is seen. This contrasts with the uneven granular staining of the cytoplasm seen with ANCA-containing serum. Moreover, preincubation of the smears with AP 268 or ANCAcontaining serum does not influence the binding of the other. Alkaline phosphatase is located at the cell membrane4 (the an
ELISA-identified binding of IgG from WG and control serum diluted 1 :80 to percoll gradient fractions of human neutrophil cytoplasm characterised by biochemical markers including alkaline phosphatase.
fractions
containing alkaline phosphatase are those containing the membranes3) but ANCA is directed against a cellular compartment with a density between that of the primary and secondary granules of the neutrophils (repeated and reproduced with nine different preparations and 14 different sera), as seen in the accompanying figure. Whether this ANCA-related antigen is located on a separate type of granule is not yet established. Since ANCA is specific for WG (provided that microscopic polyarteritiss is classified as a subgroup of WG) identification of the ANCA-related antigen may prove to be an important step in the elucidation of the pathogenesis of WG and/or microscopic polyarteritis. Our observations do not exclude the possibility that ANCA can react or be associated with alkaline phosphatase but they strongly suggest that ANCA-related antigen is not the enzyme itself. N. R. is supported by Weimann’s Legat and the Danish Arthritis Foundation (grant 233-490). N. B. is supported by the Danish Medical Research Council (grant 12-6139), the Danish Cancer Society (grant 86-132), and the Danish Arthritis Foundation (grant 233-627).
Laboratory of Autoimmune Serology, State Serum Institute, DK-2300 Copenhagen S Denmark; and Department of Medicine and Haematology C, Gentofte University Hospital, Hellerup
NIELS RASMUSSEN NIELS BORREGAARD ALLAN WIIK
der Woude FJ, Rasmussen N, Lobatto S, et al. Autoantibodies against neutrophils and monocytes: Tool for diagnosis and marker of disease activity m Wegener’s granulomatosis. Lancet 1985; i: 425-29. 2. Rasmussen N, Wiik A. Autoimmunity in Wegener’s granulomatosis. In: Veldman JE, McCabe BF, Huizing EH, Mygind N, eds. Immunobiology, autoimmunity, transplantation, in otorhinolaryngology. Amsterdam. Kugler, 1985: 207-12. 3. Borregaard N, Heiple JM, Simons ER, Clark RA. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: Translocation during activation. J Cell Biol 1983; 97: 52-61. 4. Borgers M, Thone F, de Cree J, de Cock W. Alkaline phosphatase activity in human polymorphonuclear leukocytes. Histochem J 1978; 10: 31-43. 5. Savage COS, Winearls CG, Evans DJ, Rees AJ, Lockwood CM. Microscopic polyarteritis. Presentation, pathology and prognosis. Quart J Med 1985; 56: 467-84. 1.
van
SIR,-Antibodies against cytoplasmic antigens of neutrophil granulocytes and monocytes (ACPA) are of value both for screening and for monitoring disease activity in Wegener’s granulomatosis (WG).l,2 The strong association between titre and disease activity suggests that these autoantibodies may be involved in the pathogenesis of WG. On the other hand, the fact that the target antigen is intracellular, and thus not accessible to circulating antibodies, casts doubt on the immunopathogenic relevance of ACPA in vivo. Dr Lockwood and colleagues claim that the target antigen is associated with alkaline phosphatase, which is partly located in the cytoplasmic membrane of neutrophils and is thus accessible to ACPA. However, our findings argue against the conclusion that the target antigen is alkaline phosphatase.