388
cedent infection does not further elucidate the possible role of I.C.A. in the aetiology of diabetes. Furthermore, the absence of any association between i.c.A. and the presence of thyrogastric antibodies in a juvenile-onset diabetic13 may indicate that i.c.A. production is a transient phenomenon resulting from islet-cell and is not part of an autoimmune destructive process. Our results indicate that further simultaneous prospective studies in different centres are required to confirm the possible important relations between specific viral and HLA linked factors.
injury
We thank all those paediatricians and physicians on Merseyside and in surrounding parts of Lancashire and Cheshire and Sister J. Abbot for their cooperation in making this study possible. The late Dr F. P. Hudson, Alder Hey Hospital, Liverpool, gave us much encouragement. The National Tissue Typing Reference Laboratory, Bristol, supplied HLA typmg antisera. R.L. was supported by a grant from the Medical Research Council.
Requests for reprints should be addressed to A. G. C., Dunn Laboratories, St Bartholomew’s Hospital, London EC1A 7BE. REFERENCES 1. Cudworth, A. G., Woodrow, J. C. Br. med. J. 1975, iii, 133. 2. Cudworth, A. G., Woodrow, J. C. ibid. 1976, ii, 846. 3. Bloom, A., Hayes, T. M., Gamble, D. R. ibid. 1975, iii, 580. 4. Lancet, 1976, ii, 28. 5. Gamble, D. R., Kinsley, M. L., FitzGerald, M. G., Bolton, R., Taylor, K. W. Br. med. J. 1969, iii, 627. 6. Gamble, D. R., Taylor, K. W., Cumming, H. ibid. 1973, iv, 260. 7. Lendrum, R., Walker, G., Gamble, D. R. Lancet, 1975, i, 880. 8. Terasaki, P. I., McClelland, J. D. Nature, 1964, 204, 998. 9. Taylor, C. E. D. in Progress in Microbiological Techniques (edited by C. M. Collins); p. 1. London, 1967. 10. Haldane, J. B. S. Ann. hum. Genet. 1955, 20, 309. 11. Bottazzo, G. F., Florin-Christensen, A., Doniach, D. Lancet, 1974, ii, 1279. 12. Lendrum, R., Nelson, P. G., Pyke, D. A., Walker, G., Gamble, D. R. Br.
med. J. 1976, i, 553. 13. Lendrum, R., Walker, G., Cudworth, A. G., Woodrow, J. C., Gamble, D. R.
ibid. p. 1565.
SEVERE STEROID-RESPONSIVE NEPHROSIS ASSOCIATED WITH HYPERSENSITIVITY D. H. SANDBERG C. W. BERNSTEIN
R. M. McINTOSH R. CARR
J. STRAUSS University of Miami School of Medicine, Miami, Florida, and University of Colorado Medical Center, Denver, Colorado, U.S.A.
Sensitivity to cow’s whole milk was investigated in six patients with the idiopathic nephrotic syndrome of childhood. Prednisone was discontinued and an elemental diet was given. After proteinuria had decreased to ≤500 mg/24 hours on consecutive days patients were challenged with cow’s milk. This resulted in the return of significant proteinuria, œdema, and decreased urine volume together with a decrease in serum-IgG concentrations in four patients. Acute alteration of plasma C3 complement component accompanied milk challenge in all 6 patients. There were no consistent alterations in other immunoglobulin concentrations. Intradermal skin testing with cow’s milk extract was positive in all patients. These results suggest Summary
that in some individuals clinical and biochemical manifestations of the nephrotic syndrome of childhood may be related to hypersensitivity to food products.
Introduction THE nephrotic syndrome has been observed in association with hypersensitivity reactions to insect stings, drugs, toxins, pollens, and other inhalants, as well as foods.l2 sensitivity to foods or inhalants has been suggested as being related to relapse in children with nephrosis.3-7 We have systematically examined the role of food hypersensitivity in exacerbation of frequently
relapsing steroid-responsive nephrosis. In addition, we attempted to determine the sequence of pathogenic events
role of
involved in this association and to evaluate the dietary modifications in therapy of this disease.
Patients and Methods Six patients (four males and two females), aged 10-13 years, with severe steroid-responsive nephrotic syndrome were
studied. This selection was based on a history of frequent relapses of the nephrotic syndrome requiring repeated courses of corticosteroid therapy for control of the nephrosis and in some cases use of cytotoxic agents because of complications associated with prolonged use of adrenal corticosteroids. Percutaneous renal biopsy was performed on five of the patients and tissue was studied by light microscopy, electron microscopy, and immunohistology with fluorescein-isothiocyanate-conjugated rabbit antisera to human IgM, IgG, and C3. Careful histories were obtained with emphasis on allergic symptoms. All study patients fulfilled the clinical and biochemical criteria for idiopathic nephrotic syndrome of childhood. Informed consent was obtained from all patients and their parents. Upon entering the study prednisone was discontinued. Qualitative urinary protein was tested daily with ’Albustix’ (Ames). When significant proteinuria (>2+) was detected in consecutive daily urine specimens, the patients were admitted to the Clinical Research Center of the University of Miami School of Medicine for investigation. During a baseline period and at frequent intervals thereafter, studies included the following: careful daily record of body-weight, 24-hour urine volume, and fluid intake; complete blood-count; serum creatinine, cholesterol, sodium,. potassium, chloride, carbon dioxide, urea nitrogen, K-2-macroglobulin, total protein, protein electrophoresis, C3 proactivator, immunoglobulins G, M, A, and E, Cl, C3, C4, C5, C6, and C7, properdin, and transferrin;8 urine analysis, 24-hour urine protein excretion, urinary excretion of creatinine, urea, sodium, potassium, chloride, IgG, transferrin, and urine protein electrophoresis. After the above tests were obtained, patients were placed on an elemental liquid formula as their sole diet, provided as ’Vivonex’ or ’Flexical’, 1800-2400 kcal/24 hours in six feedings. After protein excretion decreased to 500 mg/24 hours on consecutive days, patients were evaluated for sensitivity to cow’s whole milk by oral challenge and skin testing. The oral challenge method consisted in feeding 30 g cow’s whole milk, after a 12-hour fast. Blood specimens were obtained before and 1-35 and 4.00 hours after ingestion for determination of altera tion of plasma C3 complement component.9 10 Skin tests were performed with cow’s whole milk extract by means of the serial dilution intradermal testing technique described by Miller."
Results Clinical features and renal structural and immunohistological findings in the six patients studied are summarised in table i. There was clear history with evidence of allergic symptoms in five of the patients. Examination of renal tissue revealed no significant glomerular alterations in any of the five biopsy specimens studied; deposits of immunoglobulins and complement components were not detected by immunohistolo-
389 Ultrastructural studies revealed no electron-dense deposits in the glomeruli. Proteinuria developed in all patients except case 4 when prednisone was discontinued. This increased protein excretion was associated with hypercholesterol semia, increase in weight, and oedema. Serum total protein and albumin concentrations were decreased while ot2-globulins were increased. Serum-IgG concentration was reduced. Serum C3 and C4 concentrations were normal in four of five patients in whom they were measured. Serum-IgE concentrations when measured were within the normal range (table n). Proteinuria was highly selective. On an elemental diet, protein excretion decreased to less than 500 mg/24 hours. This decrease in proteinuria was observed in association with a decrease in weight and increase in urine output (fig. 1) Reversal of abnormalities occurred as early as 1 day and usually within one week after introduction of an elemental diet (mean + S. D.; 4 ±3-2 days). Laxative administration shortened this period. Challenge with milk resulted in the return of significant proteinuria, oedema, and decreased urine volume
gical studies.
(table n). This was accompanied by a decrease in serumIgG concentrations in 4 patients; there were no consistent alterations in other immunoglobulin concentrations. At no time during the course of investigation were serum-IgE concentrations raised, and there was no evidence of alternative complement pathway activation. These changes occurred as early as 1 day and usually within 3 days (mean and s.D. 2-8±1-8 days) of milk challenge. In patients 5 and 6 there were prolonged relapses lasting 63 and 59 days, respectively. Relapse ultimately was controlled by the cereal-free diet of Rowe et al. 12 and 20 mg prednisone daily. Withdrawal of milk was followed by pronounced reduction in proteinuria to less than 500 mg/24 hours and return of serum immunoglobulins and total protein concentrations toward normal. Remission occurred rapidly in cases 1, 2, and 3 (mean±s.D., 6’3±3-5days; range, 3-10 days). Oral challenge with one ounce of cow’s milk produced activation of C3 in all six patients.lO Intradermal skin testing with cow’s milk extract was positive in all patients. Results obtained from study of two patients are illustrated in figs. 1 and 2.
TABLE I--CLINICAL DATA AND BIOPSY RESULTS IN SIX PATIENTS WITH STEROID-DEPENDENT NEPHROSIS
TABLE II-LABORATORY
RESULTS, WEIGHT,
AND NET FLUID BALANCE IN PATIENTS SHOWN IN TABLE I DURING DIFFERENT
DIETARY REGIMENS
A=elemental diet; B=milk added to diet; C=mllk removed from diet. * Case 4 did not relapse during this period.
390
Discussion The
and
of the nephrotic syndrome remains unclear. Furthermore, it is uncertain whether the changes observed are a cause or a result of the nephrotic state. Since immunoglobulins and complement components have not been demonstrated in the glomeruli of patients with minimal change disease it was suggested that this disease is not secondary to an immune-complex mechanism.’ 15 Reaginic antibody (IgE) has been implicated in the pathogenesis of this disease.’6 17 This hypothesis is based on presence of raised serum-IgE concentrations in nephrosis, or on localisation of IgE in the glomeruli of some of these patients, an observation which has not been reported by other immunohistopathologists.18 19 Furthermore, raised concentrations of serum and urinary patients have not been consistently IgE in nephrotic observed .20 21 It is known that serum-IgG concentration decreases during relapse and increases toward normal concentrations in remission. The decreased concentrations are not the result of increased urinary loss.22 C3 complement in serum also decreases during relapse.23 Suggestions that hypersensitivity is involved in the pathogenesis of the nephrotic syndrome have been based mainly on reports of clinical experience, most of which were published before percutaneous renal biopsy was commonly used.12 More precise data are needed to support these observations. Our studies in a selected group of patients suggest that in some individuals the clinical and biochemical manifestations of the nephrotic syndrome of childhood may indeed be related to hypersensitivity. The exacerbations and remissions of the nephrotic syndrome associated with changes in diet strongly support the idea that food allergens may be related to the clinical activity of this disease process. Other environmental agents such as inhalant antigens or chemicals are probably also related to exacerbations and remissions of the disease. 13 Currently available therapeutic approaches to nephrosis are not without hazards and definition of Eetiological factors and pathogenic mechanisms may lead to less toxic, more definitive, and more effective approaches to treatment of this not uncommon condition in children.
pathogenesis of
aetiology lipoid nephrosis pathogenesis (idiopathic nephrotic syndrome of childhood, minimal change disease) remains unknown. Several observations have suggested a role for cell-mediated immunity ; 13 14 however, the relation of cell-mediated immunity to the
Fig. I-Changes in urine output, weight, and protein during dietary changes in a patient with nephrosis. Reg.=regular diet.
excretion
These studies were supported in part by grants from University of Miami Hospitals and Clinics, Womens Auxiliary (North Dade Chapter) ; Clinical Research Center grant RR00261, Division of Research Resources, National Institutes of Health; Miami Dolphins Wives Charity Organization through The Kidney Foundation of Dade County and the Rocky Mountain Kidney Foundation.
Requests for reprints should be addressed to D.H.S., Clinical Research Center, University of Miami School of Medicine, P.O. Box 875, Biscayne Annex, Miami, Florida 33152, U.S.A. REFERENCES
Schreiner, G. E. in Diseases of the Kidney (edited by M. B. Strauss and L. G. Welt); vol. I, p. 503. Boston, Massachusetts, 1971. 2. Fontana, V. J., Spain, W. G., DeSantis, A. New Y. St. J. Med. 1956, 56, 1.
3907.
3. Thomson, P. D., Barratt, T. M., Stokes, C. R., Turner, M. W., Soothill, J. F. Lancet, 1976, ii, 765. 4. Matsumura, T., Koroume, T. Jap. J. Pediat. 1961, 14, 921. 5. Matsumura, T., Koroume, T., Matsui, A., Fukazawa, T., Kimura, S., Tamura, H. Proc 13th Int. Cong. Pediat. 1971, p. 41. 6. Sandberg, D. H., Bernstein, C. W. McIntosh, R. M., Deling, B., Strauss, J. in Pediatric Nephrology, (edited by J. Strauss); vol. II, p. 191. New York, 1976. 7.
Fig. 2--Changes in some serums proteins and protein excretion during dietary changes in a patient with nephrosis.
D. H., McIntosh, R. M., Bernstein, C. W., Deling, B., Strauss, J. Pediat. *Res. 1975, 9, 378 (abst.). 8. McIntosh, R. M., Griswold, W. R., Chernack, W. B., Williams, G., Strauss, J., Kaufman, D. B., Koss, M. N., McIntosh, J. R., Cohen, R., Weil, R. III. Q. Jl Med. 1975, 44, 285.
Sandberg,
391
TRANSITORY DECREASE IN PLATELET MONOAMINE-OXIDASE ACTIVITY DURING MIGRAINE ATTACKS VIVETTE GLOVER
MERTON SANDLER
Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynæcology, Queen Charlotte’s Maternity Hospital, London W6 0XG
ELLEN GRANT
F. CLIFFORD ROSE
Department of Neurology, Charing Cross Hospital, London W6 8RF
MARCIA WILKINSON
D. ORTON
nificant decrease outside an attack compared with controls. In an attempt to define the biochemical lesion more clearly, we have carried out a larger survey of platelet M.A.O. activity in migrainous patients than those previously performed and we have examined platelet samples from subjects both during and outside an attack, as well as determining whether the observed effects might stem from the prior administration of antimigrainous drugs. We also charted platelet M.A.o. activity in a group of children with recurrent abdominal pain, a condition associated with an increased risk of developing migraine in adult life7and which has been designated a "migraine equivalent" by Sachs.8
Princess Margaret Migraine Clinic, London EC1M 6DX
Department of Pœdiatrics, Southmead General Hospital, Bristol BS10 5NB
A
highly significant
monoamine-oxidase
decrease in platelet activity has been
observed in migrainous subjects during a migraine attack compared with activity outside an attack. The effect did not derive from drugs commonly used in
migraine therapy. Introduction
Hanington noted that reported to initiate an attack
certain foods, commonly of migraine, contain tyramine and are similar to those causing adverse reactions in subjects taking monoamine-oxidase (M.A.o.)-inhibitory drugs. She therefore suggested that M.A.o., which inactivates tyramine, might be reduced in migrainous patients. The ability of tyramine itself to provoke an attack in dietary migraine sufferersl is still controversial. 23 Even so, evidence has emerged suggesting that platelet M.A.o. activity is decreased in this clinical group, although the suggested manner in which the impairment manifests itself has differed in the three studies published to date: the first, a pilot study,4 noted a decrease in activity during an attack, the secondshowed that enzyme activity is lower and more variable in migrainous patients compared with controls both during and outside an attack, whilst the third6 recorded a sig-
9. Matthews, T. S., Soothill, J. F. Lancet, 1970, ii, 893. 10. Sandberg, D. H., Bernstein, C. W. Pediat. Res. 1972, 6, 383. 11. Miller, J. B. Food Allergy: Provocative Testing and Neutralization Therapy. Springfield, Illinois, 1972. 12. Rowe, A. H., Rowe, A. H., Jr. Food Allergy: Its Manifestations and Control and the Elimination Diets, p. 41. Springfield, Illinois, 1972. 13. Shalhoub, R. J. Lancet, 1974, ii, 556. 14. Moorthy, A. V., Zimmerman, S. W., Burkholder, P. M. ibid. 1976, i, 1160. 15. Drummond, K. N., Michael, A. F., Good, R. A., Vernier, R. J. clin. Invest.
1966, 45, 620. 16. Gerber, M. A., Paronetto, F. Lancet, 1971, i, 1097. 17. Hamburger, R., Tune, B. J. Pediat. 1973, 83, 767. 18. Lewis, E. J., Kallen, R. J., Rowe, D. S. Lancet, 1973, i, 1395. 19. Roy, L. P., Westberg, N. G., Michael, A. F. Clin. exp. Immun. 1973, 13, 553. 20. Barratt, T. M., Turner, M. W., Johansson, S. G. O. Lancet, 1971, ii, 402. 21. Trygstad, C. W., Heiner, D. C. Pediat. Res. 1975, 9, 380. (abstr.). 22. Giangiacomo, J., Cleary, T., Cole, B. R., Hoffsten, P., Robson, A. M. New
Engl. J. Med. 1975, 293, 8. Slobody, L. B., Strang, R. H. J. clin. Invest. 1973, 32, 581.
23. Lange, K.,
study, patients were self-selected as a group of females and twenty-two males presenting during or outside an attack at the Charing Cross Hospital Migraine Clinic. A subgroup was classified as having dietary migraine, depending on the alleged ability of foods such as cheese or chocolate to initiate attacks. This patient-group had an average age of 40 years. A further small group of adult patients, three females and three males, presented with an attack at the Princess Margaret Migraine Clinic and these returned later outside an attack. Controls were chosen from laboratory and hospital staff (twenty-five females and thirteen males) without migraine who had a mean age of 36. Samples from Charing Cross patients and controls were prepared within days of each other, stored frozen, and assayed at the same time. Princess Margeret Migraine Clinic samples were prepared and assayed separately. The sixteen children with recurrent periumbilical abdominal pain, in whom no organic illness could be found, had a mean age of 8.75 years (range 5-13 years). There were nine females and seven males. Venepuncture was only performed when blood was required for routine hwmatological examination, and in no case was it obtained during an attack. A control group of twenty-one children had a similar mean age of 9.14 years (range 5-13 years) with eleven females and ten males. Blood was taken during routine hsmatological investigation before surgery. None of these control subjects had a history of recurrent abdominal pain. For the adult study, blood (10 ml) obtained by venepuncture in the morning was added immediately to a plastic ’Universal’ container containing 10 ml of 2% disodium ethylenediaminetetraacetate (Na2E.D.T.A.) in physiological saline and mixed gently but thoroughly. Red cells were allowed to settle overnight at 20°C and platelet-rich plasma aspirated off the following day. This procedure produced platelets with the same specific M.A.O. activity as those harvested within an hour of blood collection. A different technique" was used for the study on children. Platelet-rich plasma, prepared by either method, was centrifuged at 2500 g for 15 min, the supernatant discarded, and the white upper layer of the platelet button resuspended in 1 ml of 0-3mol/1 sucrose, leaving any red cells undisturbed. The resuspended platelets were centrifuged again at 2500 g for 15 min, resuspended in 1 ml of 0-3 moVI sucrose, and stored at -20°C. Each sample was frozen and thawed twice and mixed in a ’Vortex’ mixer before assay. Protein was assayed by the method of Lowry et al.10 M.A.o. was assayed by a scaleddown modification of the method of Robinson et al. 11 Platelet suspension (20 1) was preincubated for 5 min at 37°C with 100 0 of 100 mmol/1 phosphate buffer, pH 7-4. Blanks without enzyme and with boiled enzyme were incubated with each batch. The reaction was shown to proceed linearly for 30 min. The reaction was started by adding 20 fLI of l4C-tyramine, l4C-phenylethylamine, or l4C-tryptamine to the incubation mixture (final concentrations, 140, 70, and 140 mmol/1 resFor the adult
DAVID STEVENS
Summary
Subjects and Methods fifty-five
pectively). Tyramine [1-14C] (56 mCi/mmol), &bgr;-phenylethylamine-HCl (1-’4C] (9-9 mCi/mmol), and tryptamine bisuc-
cedent infection does not further elucidate the possible role of I.C.A. in the aetiology of diabetes. Furthermore, the absence of any association between i.c.A. and the presence of thyrogastric antibodies in a juvenile-onset diabetic13 may indicate that i.c.A. production is a transient phenomenon resulting from islet-cell and is not part of an autoimmune destructive process. Our results indicate that further simultaneous prospective studies in different centres are required to confirm the possible important relations between specific viral and HLA linked factors.
injury
We thank all those paediatricians and physicians on Merseyside and in surrounding parts of Lancashire and Cheshire and Sister J. Abbot for their cooperation in making this study possible. The late Dr F. P. Hudson, Alder Hey Hospital, Liverpool, gave us much encouragement. The National Tissue Typing Reference Laboratory, Bristol, supplied HLA typmg antisera. R.L. was supported by a grant from the Medical Research Council.
Requests for reprints should be addressed to A. G. C., Dunn Laboratories, St Bartholomew’s Hospital, London EC1A 7BE. REFERENCES 1. Cudworth, A. G., Woodrow, J. C. Br. med. J. 1975, iii, 133. 2. Cudworth, A. G., Woodrow, J. C. ibid. 1976, ii, 846. 3. Bloom, A., Hayes, T. M., Gamble, D. R. ibid. 1975, iii, 580. 4. Lancet, 1976, ii, 28. 5. Gamble, D. R., Kinsley, M. L., FitzGerald, M. G., Bolton, R., Taylor, K. W. Br. med. J. 1969, iii, 627. 6. Gamble, D. R., Taylor, K. W., Cumming, H. ibid. 1973, iv, 260. 7. Lendrum, R., Walker, G., Gamble, D. R. Lancet, 1975, i, 880. 8. Terasaki, P. I., McClelland, J. D. Nature, 1964, 204, 998. 9. Taylor, C. E. D. in Progress in Microbiological Techniques (edited by C. M. Collins); p. 1. London, 1967. 10. Haldane, J. B. S. Ann. hum. Genet. 1955, 20, 309. 11. Bottazzo, G. F., Florin-Christensen, A., Doniach, D. Lancet, 1974, ii, 1279. 12. Lendrum, R., Nelson, P. G., Pyke, D. A., Walker, G., Gamble, D. R. Br.
med. J. 1976, i, 553. 13. Lendrum, R., Walker, G., Cudworth, A. G., Woodrow, J. C., Gamble, D. R.
ibid. p. 1565.
SEVERE STEROID-RESPONSIVE NEPHROSIS ASSOCIATED WITH HYPERSENSITIVITY D. H. SANDBERG C. W. BERNSTEIN
R. M. McINTOSH R. CARR
J. STRAUSS University of Miami School of Medicine, Miami, Florida, and University of Colorado Medical Center, Denver, Colorado, U.S.A.
Sensitivity to cow’s whole milk was investigated in six patients with the idiopathic nephrotic syndrome of childhood. Prednisone was discontinued and an elemental diet was given. After proteinuria had decreased to ≤500 mg/24 hours on consecutive days patients were challenged with cow’s milk. This resulted in the return of significant proteinuria, œdema, and decreased urine volume together with a decrease in serum-IgG concentrations in four patients. Acute alteration of plasma C3 complement component accompanied milk challenge in all 6 patients. There were no consistent alterations in other immunoglobulin concentrations. Intradermal skin testing with cow’s milk extract was positive in all patients. These results suggest Summary
that in some individuals clinical and biochemical manifestations of the nephrotic syndrome of childhood may be related to hypersensitivity to food products.
Introduction THE nephrotic syndrome has been observed in association with hypersensitivity reactions to insect stings, drugs, toxins, pollens, and other inhalants, as well as foods.l2 sensitivity to foods or inhalants has been suggested as being related to relapse in children with nephrosis.3-7 We have systematically examined the role of food hypersensitivity in exacerbation of frequently
relapsing steroid-responsive nephrosis. In addition, we attempted to determine the sequence of pathogenic events
role of
involved in this association and to evaluate the dietary modifications in therapy of this disease.
Patients and Methods Six patients (four males and two females), aged 10-13 years, with severe steroid-responsive nephrotic syndrome were
studied. This selection was based on a history of frequent relapses of the nephrotic syndrome requiring repeated courses of corticosteroid therapy for control of the nephrosis and in some cases use of cytotoxic agents because of complications associated with prolonged use of adrenal corticosteroids. Percutaneous renal biopsy was performed on five of the patients and tissue was studied by light microscopy, electron microscopy, and immunohistology with fluorescein-isothiocyanate-conjugated rabbit antisera to human IgM, IgG, and C3. Careful histories were obtained with emphasis on allergic symptoms. All study patients fulfilled the clinical and biochemical criteria for idiopathic nephrotic syndrome of childhood. Informed consent was obtained from all patients and their parents. Upon entering the study prednisone was discontinued. Qualitative urinary protein was tested daily with ’Albustix’ (Ames). When significant proteinuria (>2+) was detected in consecutive daily urine specimens, the patients were admitted to the Clinical Research Center of the University of Miami School of Medicine for investigation. During a baseline period and at frequent intervals thereafter, studies included the following: careful daily record of body-weight, 24-hour urine volume, and fluid intake; complete blood-count; serum creatinine, cholesterol, sodium,. potassium, chloride, carbon dioxide, urea nitrogen, K-2-macroglobulin, total protein, protein electrophoresis, C3 proactivator, immunoglobulins G, M, A, and E, Cl, C3, C4, C5, C6, and C7, properdin, and transferrin;8 urine analysis, 24-hour urine protein excretion, urinary excretion of creatinine, urea, sodium, potassium, chloride, IgG, transferrin, and urine protein electrophoresis. After the above tests were obtained, patients were placed on an elemental liquid formula as their sole diet, provided as ’Vivonex’ or ’Flexical’, 1800-2400 kcal/24 hours in six feedings. After protein excretion decreased to 500 mg/24 hours on consecutive days, patients were evaluated for sensitivity to cow’s whole milk by oral challenge and skin testing. The oral challenge method consisted in feeding 30 g cow’s whole milk, after a 12-hour fast. Blood specimens were obtained before and 1-35 and 4.00 hours after ingestion for determination of altera tion of plasma C3 complement component.9 10 Skin tests were performed with cow’s whole milk extract by means of the serial dilution intradermal testing technique described by Miller."
Results Clinical features and renal structural and immunohistological findings in the six patients studied are summarised in table i. There was clear history with evidence of allergic symptoms in five of the patients. Examination of renal tissue revealed no significant glomerular alterations in any of the five biopsy specimens studied; deposits of immunoglobulins and complement components were not detected by immunohistolo-
389 Ultrastructural studies revealed no electron-dense deposits in the glomeruli. Proteinuria developed in all patients except case 4 when prednisone was discontinued. This increased protein excretion was associated with hypercholesterol semia, increase in weight, and oedema. Serum total protein and albumin concentrations were decreased while ot2-globulins were increased. Serum-IgG concentration was reduced. Serum C3 and C4 concentrations were normal in four of five patients in whom they were measured. Serum-IgE concentrations when measured were within the normal range (table n). Proteinuria was highly selective. On an elemental diet, protein excretion decreased to less than 500 mg/24 hours. This decrease in proteinuria was observed in association with a decrease in weight and increase in urine output (fig. 1) Reversal of abnormalities occurred as early as 1 day and usually within one week after introduction of an elemental diet (mean + S. D.; 4 ±3-2 days). Laxative administration shortened this period. Challenge with milk resulted in the return of significant proteinuria, oedema, and decreased urine volume
gical studies.
(table n). This was accompanied by a decrease in serumIgG concentrations in 4 patients; there were no consistent alterations in other immunoglobulin concentrations. At no time during the course of investigation were serum-IgE concentrations raised, and there was no evidence of alternative complement pathway activation. These changes occurred as early as 1 day and usually within 3 days (mean and s.D. 2-8±1-8 days) of milk challenge. In patients 5 and 6 there were prolonged relapses lasting 63 and 59 days, respectively. Relapse ultimately was controlled by the cereal-free diet of Rowe et al. 12 and 20 mg prednisone daily. Withdrawal of milk was followed by pronounced reduction in proteinuria to less than 500 mg/24 hours and return of serum immunoglobulins and total protein concentrations toward normal. Remission occurred rapidly in cases 1, 2, and 3 (mean±s.D., 6’3±3-5days; range, 3-10 days). Oral challenge with one ounce of cow’s milk produced activation of C3 in all six patients.lO Intradermal skin testing with cow’s milk extract was positive in all patients. Results obtained from study of two patients are illustrated in figs. 1 and 2.
TABLE I--CLINICAL DATA AND BIOPSY RESULTS IN SIX PATIENTS WITH STEROID-DEPENDENT NEPHROSIS
TABLE II-LABORATORY
RESULTS, WEIGHT,
AND NET FLUID BALANCE IN PATIENTS SHOWN IN TABLE I DURING DIFFERENT
DIETARY REGIMENS
A=elemental diet; B=milk added to diet; C=mllk removed from diet. * Case 4 did not relapse during this period.
390
Discussion The
and
of the nephrotic syndrome remains unclear. Furthermore, it is uncertain whether the changes observed are a cause or a result of the nephrotic state. Since immunoglobulins and complement components have not been demonstrated in the glomeruli of patients with minimal change disease it was suggested that this disease is not secondary to an immune-complex mechanism.’ 15 Reaginic antibody (IgE) has been implicated in the pathogenesis of this disease.’6 17 This hypothesis is based on presence of raised serum-IgE concentrations in nephrosis, or on localisation of IgE in the glomeruli of some of these patients, an observation which has not been reported by other immunohistopathologists.18 19 Furthermore, raised concentrations of serum and urinary patients have not been consistently IgE in nephrotic observed .20 21 It is known that serum-IgG concentration decreases during relapse and increases toward normal concentrations in remission. The decreased concentrations are not the result of increased urinary loss.22 C3 complement in serum also decreases during relapse.23 Suggestions that hypersensitivity is involved in the pathogenesis of the nephrotic syndrome have been based mainly on reports of clinical experience, most of which were published before percutaneous renal biopsy was commonly used.12 More precise data are needed to support these observations. Our studies in a selected group of patients suggest that in some individuals the clinical and biochemical manifestations of the nephrotic syndrome of childhood may indeed be related to hypersensitivity. The exacerbations and remissions of the nephrotic syndrome associated with changes in diet strongly support the idea that food allergens may be related to the clinical activity of this disease process. Other environmental agents such as inhalant antigens or chemicals are probably also related to exacerbations and remissions of the disease. 13 Currently available therapeutic approaches to nephrosis are not without hazards and definition of Eetiological factors and pathogenic mechanisms may lead to less toxic, more definitive, and more effective approaches to treatment of this not uncommon condition in children.
pathogenesis of
aetiology lipoid nephrosis pathogenesis (idiopathic nephrotic syndrome of childhood, minimal change disease) remains unknown. Several observations have suggested a role for cell-mediated immunity ; 13 14 however, the relation of cell-mediated immunity to the
Fig. I-Changes in urine output, weight, and protein during dietary changes in a patient with nephrosis. Reg.=regular diet.
excretion
These studies were supported in part by grants from University of Miami Hospitals and Clinics, Womens Auxiliary (North Dade Chapter) ; Clinical Research Center grant RR00261, Division of Research Resources, National Institutes of Health; Miami Dolphins Wives Charity Organization through The Kidney Foundation of Dade County and the Rocky Mountain Kidney Foundation.
Requests for reprints should be addressed to D.H.S., Clinical Research Center, University of Miami School of Medicine, P.O. Box 875, Biscayne Annex, Miami, Florida 33152, U.S.A. REFERENCES
Schreiner, G. E. in Diseases of the Kidney (edited by M. B. Strauss and L. G. Welt); vol. I, p. 503. Boston, Massachusetts, 1971. 2. Fontana, V. J., Spain, W. G., DeSantis, A. New Y. St. J. Med. 1956, 56, 1.
3907.
3. Thomson, P. D., Barratt, T. M., Stokes, C. R., Turner, M. W., Soothill, J. F. Lancet, 1976, ii, 765. 4. Matsumura, T., Koroume, T. Jap. J. Pediat. 1961, 14, 921. 5. Matsumura, T., Koroume, T., Matsui, A., Fukazawa, T., Kimura, S., Tamura, H. Proc 13th Int. Cong. Pediat. 1971, p. 41. 6. Sandberg, D. H., Bernstein, C. W. McIntosh, R. M., Deling, B., Strauss, J. in Pediatric Nephrology, (edited by J. Strauss); vol. II, p. 191. New York, 1976. 7.
Fig. 2--Changes in some serums proteins and protein excretion during dietary changes in a patient with nephrosis.
D. H., McIntosh, R. M., Bernstein, C. W., Deling, B., Strauss, J. Pediat. *Res. 1975, 9, 378 (abst.). 8. McIntosh, R. M., Griswold, W. R., Chernack, W. B., Williams, G., Strauss, J., Kaufman, D. B., Koss, M. N., McIntosh, J. R., Cohen, R., Weil, R. III. Q. Jl Med. 1975, 44, 285.
Sandberg,
391
TRANSITORY DECREASE IN PLATELET MONOAMINE-OXIDASE ACTIVITY DURING MIGRAINE ATTACKS VIVETTE GLOVER
MERTON SANDLER
Bernhard Baron Memorial Research Laboratories and Institute of Obstetrics and Gynæcology, Queen Charlotte’s Maternity Hospital, London W6 0XG
ELLEN GRANT
F. CLIFFORD ROSE
Department of Neurology, Charing Cross Hospital, London W6 8RF
MARCIA WILKINSON
D. ORTON
nificant decrease outside an attack compared with controls. In an attempt to define the biochemical lesion more clearly, we have carried out a larger survey of platelet M.A.O. activity in migrainous patients than those previously performed and we have examined platelet samples from subjects both during and outside an attack, as well as determining whether the observed effects might stem from the prior administration of antimigrainous drugs. We also charted platelet M.A.o. activity in a group of children with recurrent abdominal pain, a condition associated with an increased risk of developing migraine in adult life7and which has been designated a "migraine equivalent" by Sachs.8
Princess Margaret Migraine Clinic, London EC1M 6DX
Department of Pœdiatrics, Southmead General Hospital, Bristol BS10 5NB
A
highly significant
monoamine-oxidase
decrease in platelet activity has been
observed in migrainous subjects during a migraine attack compared with activity outside an attack. The effect did not derive from drugs commonly used in
migraine therapy. Introduction
Hanington noted that reported to initiate an attack
certain foods, commonly of migraine, contain tyramine and are similar to those causing adverse reactions in subjects taking monoamine-oxidase (M.A.o.)-inhibitory drugs. She therefore suggested that M.A.o., which inactivates tyramine, might be reduced in migrainous patients. The ability of tyramine itself to provoke an attack in dietary migraine sufferersl is still controversial. 23 Even so, evidence has emerged suggesting that platelet M.A.o. activity is decreased in this clinical group, although the suggested manner in which the impairment manifests itself has differed in the three studies published to date: the first, a pilot study,4 noted a decrease in activity during an attack, the secondshowed that enzyme activity is lower and more variable in migrainous patients compared with controls both during and outside an attack, whilst the third6 recorded a sig-
9. Matthews, T. S., Soothill, J. F. Lancet, 1970, ii, 893. 10. Sandberg, D. H., Bernstein, C. W. Pediat. Res. 1972, 6, 383. 11. Miller, J. B. Food Allergy: Provocative Testing and Neutralization Therapy. Springfield, Illinois, 1972. 12. Rowe, A. H., Rowe, A. H., Jr. Food Allergy: Its Manifestations and Control and the Elimination Diets, p. 41. Springfield, Illinois, 1972. 13. Shalhoub, R. J. Lancet, 1974, ii, 556. 14. Moorthy, A. V., Zimmerman, S. W., Burkholder, P. M. ibid. 1976, i, 1160. 15. Drummond, K. N., Michael, A. F., Good, R. A., Vernier, R. J. clin. Invest.
1966, 45, 620. 16. Gerber, M. A., Paronetto, F. Lancet, 1971, i, 1097. 17. Hamburger, R., Tune, B. J. Pediat. 1973, 83, 767. 18. Lewis, E. J., Kallen, R. J., Rowe, D. S. Lancet, 1973, i, 1395. 19. Roy, L. P., Westberg, N. G., Michael, A. F. Clin. exp. Immun. 1973, 13, 553. 20. Barratt, T. M., Turner, M. W., Johansson, S. G. O. Lancet, 1971, ii, 402. 21. Trygstad, C. W., Heiner, D. C. Pediat. Res. 1975, 9, 380. (abstr.). 22. Giangiacomo, J., Cleary, T., Cole, B. R., Hoffsten, P., Robson, A. M. New
Engl. J. Med. 1975, 293, 8. Slobody, L. B., Strang, R. H. J. clin. Invest. 1973, 32, 581.
23. Lange, K.,
study, patients were self-selected as a group of females and twenty-two males presenting during or outside an attack at the Charing Cross Hospital Migraine Clinic. A subgroup was classified as having dietary migraine, depending on the alleged ability of foods such as cheese or chocolate to initiate attacks. This patient-group had an average age of 40 years. A further small group of adult patients, three females and three males, presented with an attack at the Princess Margaret Migraine Clinic and these returned later outside an attack. Controls were chosen from laboratory and hospital staff (twenty-five females and thirteen males) without migraine who had a mean age of 36. Samples from Charing Cross patients and controls were prepared within days of each other, stored frozen, and assayed at the same time. Princess Margeret Migraine Clinic samples were prepared and assayed separately. The sixteen children with recurrent periumbilical abdominal pain, in whom no organic illness could be found, had a mean age of 8.75 years (range 5-13 years). There were nine females and seven males. Venepuncture was only performed when blood was required for routine hwmatological examination, and in no case was it obtained during an attack. A control group of twenty-one children had a similar mean age of 9.14 years (range 5-13 years) with eleven females and ten males. Blood was taken during routine hsmatological investigation before surgery. None of these control subjects had a history of recurrent abdominal pain. For the adult study, blood (10 ml) obtained by venepuncture in the morning was added immediately to a plastic ’Universal’ container containing 10 ml of 2% disodium ethylenediaminetetraacetate (Na2E.D.T.A.) in physiological saline and mixed gently but thoroughly. Red cells were allowed to settle overnight at 20°C and platelet-rich plasma aspirated off the following day. This procedure produced platelets with the same specific M.A.O. activity as those harvested within an hour of blood collection. A different technique" was used for the study on children. Platelet-rich plasma, prepared by either method, was centrifuged at 2500 g for 15 min, the supernatant discarded, and the white upper layer of the platelet button resuspended in 1 ml of 0-3mol/1 sucrose, leaving any red cells undisturbed. The resuspended platelets were centrifuged again at 2500 g for 15 min, resuspended in 1 ml of 0-3 moVI sucrose, and stored at -20°C. Each sample was frozen and thawed twice and mixed in a ’Vortex’ mixer before assay. Protein was assayed by the method of Lowry et al.10 M.A.o. was assayed by a scaleddown modification of the method of Robinson et al. 11 Platelet suspension (20 1) was preincubated for 5 min at 37°C with 100 0 of 100 mmol/1 phosphate buffer, pH 7-4. Blanks without enzyme and with boiled enzyme were incubated with each batch. The reaction was shown to proceed linearly for 30 min. The reaction was started by adding 20 fLI of l4C-tyramine, l4C-phenylethylamine, or l4C-tryptamine to the incubation mixture (final concentrations, 140, 70, and 140 mmol/1 resFor the adult
DAVID STEVENS
Summary
Subjects and Methods fifty-five
pectively). Tyramine [1-14C] (56 mCi/mmol), &bgr;-phenylethylamine-HCl (1-’4C] (9-9 mCi/mmol), and tryptamine bisuc-
