343
showed IgA and C3 in dermal capillary walls and connective tissues but no fibrin/fibrinogen. Concomitant deposition of IgA and C3 in dermal vessels and in glomerular mesangium was observed in one patient with H.S.P. and in the two cases with Berger’s nephritis. Clq and C4 were either not found or were present with only slight staining intensity in the dermis or glomerular mesangium of any patient. Skin from eighteen control patients was similarly studied, including that from four normal subjects, six with systemic lupus erythematosus, one with discoid lupus erythematosus, two with lipoid nephrosis, and five with other miscellaneous conditions. None of these showed staining characteristics which resembled those found in and Berger’s nephritis. Our findings indicate that dermal immunohistochemical examination is a useful diagnostic aid in patients with H.s.P. and Berger’s nephritis. The predominance of IgA/C3 immune complexes in the absence of Clq and C4 provides indirect evidence of activation of the complement system through the alternate pathway in the dermal and glomerular lesions. These parallel findings in the two syndromes suggest that they may be related. H.s.P.
Departments of Pathology, Pediatrics, and Medicine, St. Louis University School of Medicine, 1402 South Grand, St. Louis, Missouri 63104, U.S.A.
CHENG C. TSAI
JOSEPH GIANGIACOMO JACK ZUCKNER.
One digoxin tablet (0-25 mg.’Digoxin Novum ’, Star Ltd., Tampere, Finland) or 10 ml. of digoxin standard solution (0-025 mg. per ml. World Health Organisation Centre for Chemical Reference Substances, Solna, Sweden) was given with 100 ml. of water to eight healthy volunteer female subjects during a 10-day crossover trial under laboratory supervision. Venous blood-samples were taken for radioimmunological measurement of the basal level of serum-digoxin on the 8ih, 9th, and 10th morning just before the next dose of drug. After the final dosage the samples were drawn at , 1, 2, 4, 8, and 24 hours to determine the area under the absorption curve.
During
treatment
with tablets the
mean
basal digoxin
ng. per ml. of plasma) of the concentration attained with the standard solution (0-62 ±0-04 ng. per ml, of plasma). After the final dosage of digoxin tablets the area under the absorption curve was 95-0 ±9-1% (0-18±0-03 ng. per ml. per hour) of that obtained with the standard solution (0-19 ±0-02 ng. per ml. per hour). When 12 tablets were individually dissolved according to the method in the U.S.P. (U.S.P. XVIII, sixth interim revisions effective as of Nov. 15, 1973), the mean digoxin was
95-2±10-6% (s.E.) (0-59±0-05
’
1.
2. 3.
4. 5. 6.
Our was similar to that from standard solution. results accord with those of Klink et al.,6 who found that there is not always a simple correlation between the solubility and bioavailability of digoxin tablets. The results also suggest that the solution test in the U.S.P. is an unsatisfactory method of evaluating the bioavailability of commercial digoxin tablets.
tablets
Institute of Biomedical Sciences, University of Tampere, Teiskontie 35, SF-33520, Tampere 52, Finland.
PAULI YLITALO.
Research Laboratory of Star Ltd., Pinninkatu 53, SF-33100, Tampere 10, Finland.
GUNILLA WILÉN STIG LUNDELL.
HYPOKALÆMIA AND DIGOXIN-INDUCED
DISCREPANCY BETWEEN SOLUTION AND BIOAVAILABILITY OF DIGOXIN TABLETS SIR,-Many have reported an association between the solution-rate of digoxin tablets in vitro and their biological activity.’-’ However, Klink et al. found that despite different rates of solution the bioavailability of various digoxin tablets can be identical.We have found that the solution test given in the United States Pharmacopeia (U.S.P.) is not a satisfactory method of evaluating the biological activity of digoxin tablets.
level
concentration in the test solvent corresponded to only 57’8J1’6% of the declared amount, although the recovery of digoxin in these test conditions was 98-40-7% (p < 0-0005). Although the tablets tested did not meet U.S.P. specification regarding solubility (4/12 tablets dissolved less than 55%), gastrointestinal absorption of digoxin from the
Johnson, B. F., Greer, H., McCrerie, J., Bye, C., Fowle, A. Lancet, 1973, i, 1473. Lindenbaum, J., Butler, V. P., Jr., Murphy, J. E., Cresswell, R. M. ibid. p. 1215. Wagner, J. G., Christensen, M., Sakmar, E., Blair, D., Yates, J. D., Willis, P. W., III, Sedman, A. J., Stoll, R. G. J. Am. med. Ass. 1973, 224, 199. Binnion, P. F. Clin. Pharmac. Ther. 1974, 16, 807. Greenblatt, D. J., Duhme, D. W., Koch-Weser, J., Smith, T. W. ibid. 1974, 229, 1774. Klink, P. R., Poust, R. I., Colaizzi, J. L., McDonald, R. H., Jr. J. Pharm. Sci. 1974, 63, 1231.
ARRHYTHMIAS SIR,-One of the main reasons for giving potassium supplements to patients receiving diuretics and digitalis is to prevent possible hypokalaemia, which is considered to sensitise the patient to the arrhythmogenic effects of digitalis. We teach our medical students that digitalis
produces arrhythmias more readily in hypokalmmic patients than in patients in normal potassium balance, and this is supported by statements in major textbooks.7-9 What is the evidence that this is true ? Before 1952, the idea that potassium loss sensitises the myocardium to digitalis was based on clinical statements that increased sensitivity to digitalis occurred in patients on diuretic therapy in the presence of a normal serumpotassium level.lo Acetyl strophanthidin does produce rhythm disturbances at a lower dose after the acute reduction of serum-potassium concentration by haemodialysis in dogs."Similar results were produced by a glucose-insulin infusion which acutely reduces the plasma-potassium concentration, but this effect was not noted after chronic potassium depletion in dogs.12 Acute changes in the relationship between extracellular and intracellular potassium concentrations certainly affect the ability of the myocardium to take up circulating digoxin.13-17 Presumably for ventricular tachycardia (and other arrhythmias) to be produced by digoxin, a certain myocardial level has to be achieved,18 but at the time of a toxic digitalis-induced ventricular tachycardia in normal and acutely hypokalasmic dogs there is no difference in the myocardial concentration of digoxin.19-20 However, much of this work is concerned with the re-
8.
Bellet, S. Clinical Disorders of the Heart Beat; p. 1065. Philadelphia, 1971. Hudson, R. E. B. Cardiovascular Pathology; vol. 3, p. S238. Balti-
9.
Conn, H. L., Horwitz, O. Cardiac and Vascular Diseases; vol.
7.
more, 1970.
I,
Philadelphia, 1971. Lown, B., Levine, S. A. Current Concepts in Digitalis Therapy; chap. 5. Boston, 1954. Lown, B. et al. J. clin. Invest. 1952, 31, 648. Kleiger, R. E., Vitale, J., Lown, B. Proc. New Engl. cardiovasc. Soc. p. 553.
10. 11. 12.
1964-65, 23, 19. 13. Cohn, K. E., Kleiger, R. E., Harrison, D. C. Circulation Res. 1967, 20, 473. 14. Marcus, F. I., Kapadia, G. G., Goldsmith, C. J. Pharmac. Exp. Ther. 1969, 165, 136. 15. Morgan, L. M., Binnion, P. F. Cardiovasc. Res. 1970, 4, 235. 16. Binnion, P. F., Morgan, L. M. ibid. 1971, 5, 431. 17. Prindle, K. H., et al. Circulation Res. 1971, 28, 337. 18. Binnion, P. F. in Symposium on Digitalis; p. 254. Oslo, 1973. 19. Goldsmith, C. et al. Circulation, 1969, 39, III-92. 20. Binnion, P. F., Das Gupta, R. ibid. 1974, III-215.
344
lationship between acute reduction in circulating potassium levels and digitalis-induced arrhythmias, and the only discovered work on chronic potassium depletion in laboratory animals did not show increased sensitivity to the action of digitalis.12 Therefore, on what grounds are we entitled to teach that diuretic-induced chronic hypokalaemia in patients makes them more likely to have arrhythmias when digitalis is being given concomitantly ? Some recent experimental work helps delineate the problem. Dogs anaesthetised with sodium pentobarbitone were infused with 3H-digoxin at 20 tJ.g. per minute until atrioventricular dissociation occurred. This was chosen in preference to ventricular tachycardia, for in the presence of low plasma-potassium concentrations ventricular tachycardia is difficult to diagnose. Routine electrocardiograms and also atrial electrograms were done during the infusion of digoxin to help in the diagnosis of the arrhythmias, and plasma and tissue were taken for analysis for tritium content ten minutes after the digoxin infusion had been stopped after the first appearance of atrioventricular dissociation lasting more than 3 seconds. The control series contained 8 dogs, an acutely hypokalaemic group (glucoseinsulin infusion) had 10 dogs in it, while the chronic hypokalxmic dogs (hydrochlorthiazide plus high sodium, no potassium synthetic diet) contained 6 dogs. Although complete analysis has not been accomplished, the average plasma-potassium concentrations in these groups when the digoxin infusion was started were 4-0,2-3, and 2-6 meq. The amount of digoxin infused to produce per litre. atrioventricular dissociation was 151, 144, and 145 tJ.g. per kg. Digitalis-induced ventricular irritability appears to be induced first in the left ventricle,21 and the left ventricular myocardial digoxin content in the normal and acutely hypokalaemic groups (chronic hypokalasmia not yet analysed) at atrioventricular dissociation averaged 630 and 679 ng. per g. wet
weight.
In these experiments no evidence has come to light to support the idea that hypokalaemia, either acute or chronic, sensitises the myocardium to digoxin. Of particular importance are the chronic hypokalaemia experiments where 100 mg. hydrochlorthiazide a day and a zero potassium intake produced an average negative potassium balance of 78 meq. over a period of about ten days. This design was employed to give the nearest practical approximation to the condition of a patient on chronic diuretic therapy. As there was no evidence that those animals were more sensitive to digoxin, perhaps our teaching should be less
pragmatic. Section of Cardiology, Pennsylvania Hospital, Philadelphia, Pennsylvania 19107, U.S.A.
P. F. BINNION.
MULTIPLE SCLEROSIS SIR,—The paper by Sir Charles Symonds (Jan. 18, p. 155) raises some interesting points in comparative pathology and biochemistry. It also points out how sound scientific observations can be largely ignored in the quest for fashionable explanations of disease phenomena. Certainly Sir Charles Symonds is right to question Acheson’s statement that any relation between swayback and multiple sclerosis can be discounted. The demyelinating disease swayback is not a simple manifestation of copper deficiency. Copper and vitamin-A metabolism are closely interrelated and a decrease in bloodcopper level diminishes the liver’s capacity to store the vitamin in preparation for its many metabolic roles.22 Among these is the activation of sulphate to 3’-phospho21. 22.
Kastor, J. A., Spear, J. F., Moore, E. N. ibid. 1972, 45, 952. Owen, E. C. Fd Cosmet. Toxicol. 1965, 3, 701.
adenosine-5’-phosphosulphate, an essential step in the of the sulphatides of the medullary sheath. synthesis Owen 23 has discussed mechanisms by which copper and/or vitamin-A deficiency may interfere with sulphate metabolism and myelin integrity. Irving and Richards 2’ studied the relationship between the level of vitamin A in the diet and the incidence of demyelinated fibres in the medulla of the rat brain. Demyelination occurred at levels none of which was so low that overt signs of vitamin deficiency were produced. However, some consider that any damage to the nervous system in hypovitaminosis A is caused by skeletal dysplasia.25 Since the optimum human requirement of vitamin A is not known with any certainty, certain members of the community may be deficient in it. In his classic Nutrition and Disease, Mellanby suggested that a vitamin-A-rich diet might be of value in treating the inflammatory stage which occurs before the glial overgrowth in multiple sclerosis and produced some clinical evidence to support thiS.26 This work has never been adequately followed up and it is only recently that attention has once again been focused on nutritional influences in multiple sclerosis. 27 Many factors may operate in the development of this disease and the nutritional status of the individual may control how he responds to some of these-e.g., viral infection-and should not be neglected. As Mellanby remarked forty years ago, it is important that "... no possibility is missed of adding knowledge to the eetiology or therapeutics of an otherwise intractable disease ". Metabolic Unit, University College Hospital, London WC1E 6JJ.
J. GODFREY HEATHCOTE.
AGGREGATION OF HUMAN PLATELETS BY PLATELET-ACTIVATING FACTOR SIR,—Platelet-activating factor (P.A.F.) is a basic phospholipid from human or rabbit basophils which aggregates rabbit platelets and releases their vasoactive amines 28; rabbit P.A.F. has been implicated in deposition of immune complexes in acute serum sickness.29 The possible role of P.A.F. in human immunological diseases raises the question of its target cell. We have demonstrated its action on human platelets. Human platelets were prepared by centrifugation and washing on a cushion of red blood-cells.3O Aggregation was measured in an aggregometer; the heights of the recorded curves were expressed as arbitrary units. After aggregation, platelets were centrifuged and the serotonin content of supernatants and pellets was measured.31 The percentage of serotonin released into the supernatants was calculated. Human P.A.F. used in these experiments was a phospholipidic fraction from a silica-gel thin-layer chromatography. Typical results are given in the accompanying table. P.A.F. induced a two-stage aggregation curve; the second stage was most probably related to endogenous adenosine diphosphate (A.D.P.) release by the platelets; it was suppressed by apyrase, an A.D.P. inhibitor. A.D.P. itself induced first-stage and second-stage aggregations of the same magnitude as those observed with P.A.F. Clotting of 23. 24. 25. 26. 27.
28. 29. 30. 31.
Owen, E. C. Wld Rev. Nutr. Dietet. 1965, 5, 184. Irving, J. T., Richards, M. B. Biochem. J. 1940, 34, 198. McIlwain, H., Bachelard, H. S. Biochemistry and the C.N.S.; p. 300. London, 1971. Mellanby, E. Nutrition and Disease. Edinburgh, 1934. Millar, J. H. D., et al. Br. med. J. 1973, i, 765. Benveniste, J. Nature, 1974, 241, 581. Benveniste, J., Henson, P. M., Cochrane, C. G. J. exp. Med. 1972, 136, 1356. Bang, N. V., Heidenreich, R. O., Trygstad, C. W. Ann. N. Y. Acad. Sci. 1972, 201, 280. Weissbach, H., Andrews, E., Seligson, H. T. Standard Methods of Clinical Chemistry; vol. IV, p. 197. New York, 1963.
showed IgA and C3 in dermal capillary walls and connective tissues but no fibrin/fibrinogen. Concomitant deposition of IgA and C3 in dermal vessels and in glomerular mesangium was observed in one patient with H.S.P. and in the two cases with Berger’s nephritis. Clq and C4 were either not found or were present with only slight staining intensity in the dermis or glomerular mesangium of any patient. Skin from eighteen control patients was similarly studied, including that from four normal subjects, six with systemic lupus erythematosus, one with discoid lupus erythematosus, two with lipoid nephrosis, and five with other miscellaneous conditions. None of these showed staining characteristics which resembled those found in and Berger’s nephritis. Our findings indicate that dermal immunohistochemical examination is a useful diagnostic aid in patients with H.s.P. and Berger’s nephritis. The predominance of IgA/C3 immune complexes in the absence of Clq and C4 provides indirect evidence of activation of the complement system through the alternate pathway in the dermal and glomerular lesions. These parallel findings in the two syndromes suggest that they may be related. H.s.P.
Departments of Pathology, Pediatrics, and Medicine, St. Louis University School of Medicine, 1402 South Grand, St. Louis, Missouri 63104, U.S.A.
CHENG C. TSAI
JOSEPH GIANGIACOMO JACK ZUCKNER.
One digoxin tablet (0-25 mg.’Digoxin Novum ’, Star Ltd., Tampere, Finland) or 10 ml. of digoxin standard solution (0-025 mg. per ml. World Health Organisation Centre for Chemical Reference Substances, Solna, Sweden) was given with 100 ml. of water to eight healthy volunteer female subjects during a 10-day crossover trial under laboratory supervision. Venous blood-samples were taken for radioimmunological measurement of the basal level of serum-digoxin on the 8ih, 9th, and 10th morning just before the next dose of drug. After the final dosage the samples were drawn at , 1, 2, 4, 8, and 24 hours to determine the area under the absorption curve.
During
treatment
with tablets the
mean
basal digoxin
ng. per ml. of plasma) of the concentration attained with the standard solution (0-62 ±0-04 ng. per ml, of plasma). After the final dosage of digoxin tablets the area under the absorption curve was 95-0 ±9-1% (0-18±0-03 ng. per ml. per hour) of that obtained with the standard solution (0-19 ±0-02 ng. per ml. per hour). When 12 tablets were individually dissolved according to the method in the U.S.P. (U.S.P. XVIII, sixth interim revisions effective as of Nov. 15, 1973), the mean digoxin was
95-2±10-6% (s.E.) (0-59±0-05
’
1.
2. 3.
4. 5. 6.
Our was similar to that from standard solution. results accord with those of Klink et al.,6 who found that there is not always a simple correlation between the solubility and bioavailability of digoxin tablets. The results also suggest that the solution test in the U.S.P. is an unsatisfactory method of evaluating the bioavailability of commercial digoxin tablets.
tablets
Institute of Biomedical Sciences, University of Tampere, Teiskontie 35, SF-33520, Tampere 52, Finland.
PAULI YLITALO.
Research Laboratory of Star Ltd., Pinninkatu 53, SF-33100, Tampere 10, Finland.
GUNILLA WILÉN STIG LUNDELL.
HYPOKALÆMIA AND DIGOXIN-INDUCED
DISCREPANCY BETWEEN SOLUTION AND BIOAVAILABILITY OF DIGOXIN TABLETS SIR,-Many have reported an association between the solution-rate of digoxin tablets in vitro and their biological activity.’-’ However, Klink et al. found that despite different rates of solution the bioavailability of various digoxin tablets can be identical.We have found that the solution test given in the United States Pharmacopeia (U.S.P.) is not a satisfactory method of evaluating the biological activity of digoxin tablets.
level
concentration in the test solvent corresponded to only 57’8J1’6% of the declared amount, although the recovery of digoxin in these test conditions was 98-40-7% (p < 0-0005). Although the tablets tested did not meet U.S.P. specification regarding solubility (4/12 tablets dissolved less than 55%), gastrointestinal absorption of digoxin from the
Johnson, B. F., Greer, H., McCrerie, J., Bye, C., Fowle, A. Lancet, 1973, i, 1473. Lindenbaum, J., Butler, V. P., Jr., Murphy, J. E., Cresswell, R. M. ibid. p. 1215. Wagner, J. G., Christensen, M., Sakmar, E., Blair, D., Yates, J. D., Willis, P. W., III, Sedman, A. J., Stoll, R. G. J. Am. med. Ass. 1973, 224, 199. Binnion, P. F. Clin. Pharmac. Ther. 1974, 16, 807. Greenblatt, D. J., Duhme, D. W., Koch-Weser, J., Smith, T. W. ibid. 1974, 229, 1774. Klink, P. R., Poust, R. I., Colaizzi, J. L., McDonald, R. H., Jr. J. Pharm. Sci. 1974, 63, 1231.
ARRHYTHMIAS SIR,-One of the main reasons for giving potassium supplements to patients receiving diuretics and digitalis is to prevent possible hypokalaemia, which is considered to sensitise the patient to the arrhythmogenic effects of digitalis. We teach our medical students that digitalis
produces arrhythmias more readily in hypokalmmic patients than in patients in normal potassium balance, and this is supported by statements in major textbooks.7-9 What is the evidence that this is true ? Before 1952, the idea that potassium loss sensitises the myocardium to digitalis was based on clinical statements that increased sensitivity to digitalis occurred in patients on diuretic therapy in the presence of a normal serumpotassium level.lo Acetyl strophanthidin does produce rhythm disturbances at a lower dose after the acute reduction of serum-potassium concentration by haemodialysis in dogs."Similar results were produced by a glucose-insulin infusion which acutely reduces the plasma-potassium concentration, but this effect was not noted after chronic potassium depletion in dogs.12 Acute changes in the relationship between extracellular and intracellular potassium concentrations certainly affect the ability of the myocardium to take up circulating digoxin.13-17 Presumably for ventricular tachycardia (and other arrhythmias) to be produced by digoxin, a certain myocardial level has to be achieved,18 but at the time of a toxic digitalis-induced ventricular tachycardia in normal and acutely hypokalasmic dogs there is no difference in the myocardial concentration of digoxin.19-20 However, much of this work is concerned with the re-
8.
Bellet, S. Clinical Disorders of the Heart Beat; p. 1065. Philadelphia, 1971. Hudson, R. E. B. Cardiovascular Pathology; vol. 3, p. S238. Balti-
9.
Conn, H. L., Horwitz, O. Cardiac and Vascular Diseases; vol.
7.
more, 1970.
I,
Philadelphia, 1971. Lown, B., Levine, S. A. Current Concepts in Digitalis Therapy; chap. 5. Boston, 1954. Lown, B. et al. J. clin. Invest. 1952, 31, 648. Kleiger, R. E., Vitale, J., Lown, B. Proc. New Engl. cardiovasc. Soc. p. 553.
10. 11. 12.
1964-65, 23, 19. 13. Cohn, K. E., Kleiger, R. E., Harrison, D. C. Circulation Res. 1967, 20, 473. 14. Marcus, F. I., Kapadia, G. G., Goldsmith, C. J. Pharmac. Exp. Ther. 1969, 165, 136. 15. Morgan, L. M., Binnion, P. F. Cardiovasc. Res. 1970, 4, 235. 16. Binnion, P. F., Morgan, L. M. ibid. 1971, 5, 431. 17. Prindle, K. H., et al. Circulation Res. 1971, 28, 337. 18. Binnion, P. F. in Symposium on Digitalis; p. 254. Oslo, 1973. 19. Goldsmith, C. et al. Circulation, 1969, 39, III-92. 20. Binnion, P. F., Das Gupta, R. ibid. 1974, III-215.
344
lationship between acute reduction in circulating potassium levels and digitalis-induced arrhythmias, and the only discovered work on chronic potassium depletion in laboratory animals did not show increased sensitivity to the action of digitalis.12 Therefore, on what grounds are we entitled to teach that diuretic-induced chronic hypokalaemia in patients makes them more likely to have arrhythmias when digitalis is being given concomitantly ? Some recent experimental work helps delineate the problem. Dogs anaesthetised with sodium pentobarbitone were infused with 3H-digoxin at 20 tJ.g. per minute until atrioventricular dissociation occurred. This was chosen in preference to ventricular tachycardia, for in the presence of low plasma-potassium concentrations ventricular tachycardia is difficult to diagnose. Routine electrocardiograms and also atrial electrograms were done during the infusion of digoxin to help in the diagnosis of the arrhythmias, and plasma and tissue were taken for analysis for tritium content ten minutes after the digoxin infusion had been stopped after the first appearance of atrioventricular dissociation lasting more than 3 seconds. The control series contained 8 dogs, an acutely hypokalaemic group (glucoseinsulin infusion) had 10 dogs in it, while the chronic hypokalxmic dogs (hydrochlorthiazide plus high sodium, no potassium synthetic diet) contained 6 dogs. Although complete analysis has not been accomplished, the average plasma-potassium concentrations in these groups when the digoxin infusion was started were 4-0,2-3, and 2-6 meq. The amount of digoxin infused to produce per litre. atrioventricular dissociation was 151, 144, and 145 tJ.g. per kg. Digitalis-induced ventricular irritability appears to be induced first in the left ventricle,21 and the left ventricular myocardial digoxin content in the normal and acutely hypokalaemic groups (chronic hypokalasmia not yet analysed) at atrioventricular dissociation averaged 630 and 679 ng. per g. wet
weight.
In these experiments no evidence has come to light to support the idea that hypokalaemia, either acute or chronic, sensitises the myocardium to digoxin. Of particular importance are the chronic hypokalaemia experiments where 100 mg. hydrochlorthiazide a day and a zero potassium intake produced an average negative potassium balance of 78 meq. over a period of about ten days. This design was employed to give the nearest practical approximation to the condition of a patient on chronic diuretic therapy. As there was no evidence that those animals were more sensitive to digoxin, perhaps our teaching should be less
pragmatic. Section of Cardiology, Pennsylvania Hospital, Philadelphia, Pennsylvania 19107, U.S.A.
P. F. BINNION.
MULTIPLE SCLEROSIS SIR,—The paper by Sir Charles Symonds (Jan. 18, p. 155) raises some interesting points in comparative pathology and biochemistry. It also points out how sound scientific observations can be largely ignored in the quest for fashionable explanations of disease phenomena. Certainly Sir Charles Symonds is right to question Acheson’s statement that any relation between swayback and multiple sclerosis can be discounted. The demyelinating disease swayback is not a simple manifestation of copper deficiency. Copper and vitamin-A metabolism are closely interrelated and a decrease in bloodcopper level diminishes the liver’s capacity to store the vitamin in preparation for its many metabolic roles.22 Among these is the activation of sulphate to 3’-phospho21. 22.
Kastor, J. A., Spear, J. F., Moore, E. N. ibid. 1972, 45, 952. Owen, E. C. Fd Cosmet. Toxicol. 1965, 3, 701.
adenosine-5’-phosphosulphate, an essential step in the of the sulphatides of the medullary sheath. synthesis Owen 23 has discussed mechanisms by which copper and/or vitamin-A deficiency may interfere with sulphate metabolism and myelin integrity. Irving and Richards 2’ studied the relationship between the level of vitamin A in the diet and the incidence of demyelinated fibres in the medulla of the rat brain. Demyelination occurred at levels none of which was so low that overt signs of vitamin deficiency were produced. However, some consider that any damage to the nervous system in hypovitaminosis A is caused by skeletal dysplasia.25 Since the optimum human requirement of vitamin A is not known with any certainty, certain members of the community may be deficient in it. In his classic Nutrition and Disease, Mellanby suggested that a vitamin-A-rich diet might be of value in treating the inflammatory stage which occurs before the glial overgrowth in multiple sclerosis and produced some clinical evidence to support thiS.26 This work has never been adequately followed up and it is only recently that attention has once again been focused on nutritional influences in multiple sclerosis. 27 Many factors may operate in the development of this disease and the nutritional status of the individual may control how he responds to some of these-e.g., viral infection-and should not be neglected. As Mellanby remarked forty years ago, it is important that "... no possibility is missed of adding knowledge to the eetiology or therapeutics of an otherwise intractable disease ". Metabolic Unit, University College Hospital, London WC1E 6JJ.
J. GODFREY HEATHCOTE.
AGGREGATION OF HUMAN PLATELETS BY PLATELET-ACTIVATING FACTOR SIR,—Platelet-activating factor (P.A.F.) is a basic phospholipid from human or rabbit basophils which aggregates rabbit platelets and releases their vasoactive amines 28; rabbit P.A.F. has been implicated in deposition of immune complexes in acute serum sickness.29 The possible role of P.A.F. in human immunological diseases raises the question of its target cell. We have demonstrated its action on human platelets. Human platelets were prepared by centrifugation and washing on a cushion of red blood-cells.3O Aggregation was measured in an aggregometer; the heights of the recorded curves were expressed as arbitrary units. After aggregation, platelets were centrifuged and the serotonin content of supernatants and pellets was measured.31 The percentage of serotonin released into the supernatants was calculated. Human P.A.F. used in these experiments was a phospholipidic fraction from a silica-gel thin-layer chromatography. Typical results are given in the accompanying table. P.A.F. induced a two-stage aggregation curve; the second stage was most probably related to endogenous adenosine diphosphate (A.D.P.) release by the platelets; it was suppressed by apyrase, an A.D.P. inhibitor. A.D.P. itself induced first-stage and second-stage aggregations of the same magnitude as those observed with P.A.F. Clotting of 23. 24. 25. 26. 27.
28. 29. 30. 31.
Owen, E. C. Wld Rev. Nutr. Dietet. 1965, 5, 184. Irving, J. T., Richards, M. B. Biochem. J. 1940, 34, 198. McIlwain, H., Bachelard, H. S. Biochemistry and the C.N.S.; p. 300. London, 1971. Mellanby, E. Nutrition and Disease. Edinburgh, 1934. Millar, J. H. D., et al. Br. med. J. 1973, i, 765. Benveniste, J. Nature, 1974, 241, 581. Benveniste, J., Henson, P. M., Cochrane, C. G. J. exp. Med. 1972, 136, 1356. Bang, N. V., Heidenreich, R. O., Trygstad, C. W. Ann. N. Y. Acad. Sci. 1972, 201, 280. Weissbach, H., Andrews, E., Seligson, H. T. Standard Methods of Clinical Chemistry; vol. IV, p. 197. New York, 1963.
