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-
392 cinate
[side-chain 2-’4C] (47 mCi/mmol), obtained from New England Nuclear Corporation, had been diluted with 0.005
PLATELET
(M.A.O.)
ACTIVITY
PROTEIN/30 MIN) IN
(nmol TYRAMINE OXIDISED/mg
MIGRAINE SUFFERERS AND CONTROLS
to activities of 2-5, 2.5, and 10 µCi/µmol, respectively, and stored frozen in 0.2 ml batches. The tyramine and tryptamine were diluted 1 in 5 and the phenylethylamine 1 in 10 with water just before use. After incubation at 37°C for 30 min in a shaking water bath, the reactions were terminated by placing the tubes on ice and adding 0.4 ml of cold phosphate buffer to each. The contents of the tubes were then transferred separately to 3 cm columns of prepared ’Amberlite CG 50’ (British Drug Houses, chromatographic grade, type 11,200 mesh), and each was allowed to drain into a scintillation vial. Columns were washed with 2 ml of water and the total eluate counted with 10 ml of ’Instagel’ (Packard Instrument Ltd.).
mol/l HCI and unlabelled substrate
Results Mean platelet M.A.o. activity in the migraine group as whole was significantly lower than that of the control group (fig. 1A). The migraine group was subdivided into patients examined during and outside an attack; the former showed much lower activity than the control group while activity values in the latter, although lower, were not significantly so. Values were significantly lower during an attack than outside it (fig. 1B). There was no difference in activity outside an attack between patients with dietary and non-dietary migraine (table I). Platelet M.A.o. activity was higher in women than men, whether migrainous or control (table i). 9 Charing Cross patients, presenting during an attack, returned for a second blood-test outside an attack; their platelet activity was significantly lower during the headache episode (fig. 2), 10.9±1.54 compared a
Fig. 1—M.A.O. activity (meanS. E.) in platelet samples: (A) from migrainous subjects, without regard for phase of illness, compared with control group; (B) obtained during attack compared with outside attack and compared with control group. A, p
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-
392 cinate
[side-chain 2-’4C] (47 mCi/mmol), obtained from New England Nuclear Corporation, had been diluted with 0.005
PLATELET
(M.A.O.)
ACTIVITY
PROTEIN/30 MIN) IN
(nmol TYRAMINE OXIDISED/mg
MIGRAINE SUFFERERS AND CONTROLS
to activities of 2-5, 2.5, and 10 µCi/µmol, respectively, and stored frozen in 0.2 ml batches. The tyramine and tryptamine were diluted 1 in 5 and the phenylethylamine 1 in 10 with water just before use. After incubation at 37°C for 30 min in a shaking water bath, the reactions were terminated by placing the tubes on ice and adding 0.4 ml of cold phosphate buffer to each. The contents of the tubes were then transferred separately to 3 cm columns of prepared ’Amberlite CG 50’ (British Drug Houses, chromatographic grade, type 11,200 mesh), and each was allowed to drain into a scintillation vial. Columns were washed with 2 ml of water and the total eluate counted with 10 ml of ’Instagel’ (Packard Instrument Ltd.).
mol/l HCI and unlabelled substrate
Results Mean platelet M.A.o. activity in the migraine group as whole was significantly lower than that of the control group (fig. 1A). The migraine group was subdivided into patients examined during and outside an attack; the former showed much lower activity than the control group while activity values in the latter, although lower, were not significantly so. Values were significantly lower during an attack than outside it (fig. 1B). There was no difference in activity outside an attack between patients with dietary and non-dietary migraine (table I). Platelet M.A.o. activity was higher in women than men, whether migrainous or control (table i). 9 Charing Cross patients, presenting during an attack, returned for a second blood-test outside an attack; their platelet activity was significantly lower during the headache episode (fig. 2), 10.9±1.54 compared a
Fig. 1—M.A.O. activity (meanS. E.) in platelet samples: (A) from migrainous subjects, without regard for phase of illness, compared with control group; (B) obtained during attack compared with outside attack and compared with control group. A, p
