TOXICITY OF FOLIC ACID GIVEN IN PHARMACOLOGICAL DOSES TO HEALTHY VOLUNTEERS

61 Glibenclamide tablets

Requests for reprints

were supplied by Roussel Laboratories. should be addressed to P. B.

REFERENCES

Loubatières, A., Mariani, M. M., Ribes, G., de Malbosc, H., Chapal, J. Diabetologia, 1969, 5, 1. 2. Mehnert, H., Karg, E. Dt. med. Wschr. 1969, 16, 819. 3. Middleton, J. E., Griffiths, W. J. Br. med. J. 1957, ii, 1525. 4. Morgan, C. R., Lazarow, A. Diabetes, 1963, 12, 115. 5. Hartog, M., Gafaar, M. A., Meisser, B., Fraser, R. Br. med. J. 1964, ii, 1229. 6. Mattingly, D. J. clin. Path. 1962, 15, 374. 7. Scholz, R., Schmitz, H., Bucher, Th., Lampen, J. O. Biochim. Z. 1959, 331, 71. 8. Gloster, J. A., Harris, P. Clin. Chim. Acta, 1962, 7, 206. 9. Bernard, A. G., Ginsburg, J. Br. med. J. 1965, i, 1437. 10. de Wardener, H. E. The Kidney. London, 1967. 11. Christ, O. E., Hertner, W., Rupp, W. Horm. metab. Res. 1969, suppl. no. 1, p. 51. 12. Kraegen, E. W., Simons, R., Young, J. D. Aust. J. med. Sci. 1968, 30, 417. 13. Fussganger, R. D., Goberna, R., Hinz, M., Jaros, P., Karsten, C., Pfeiffer, E. F., Raptis, S. Horm. metab. Res. 1969, suppl. no. 1, p. 34. 14. Raptis, S., Rau, R. M., Schroder, K. E., Faulhaber, J. D., Pfeiffer, E. F. ibid. p. 65. 1.

Fig. 5-Effect of an oral glucose load on mean blood-glucose in a healthy male before and 3 hours after 5 mg. glibenclamide.

sulphonylurea is illustrated by the fact that satisfactory diabetic control could be established with as little as 2-5 mg. daily compared with 500 mg. chlorpropamide or 1000 mg. tolbutamide. When patients were changed from other sulphonylureas to glibenclamide, improved control was evident in some of the twenty-five patients previously receiving maximum doses of chlorpropamide or tolbutamide alone. In three out of sixteen of these patients, previously poor control became satisfactory on changing to glibenclamide and ten out of the twenty-five showed improved control on the new drug. Substitution of glibenclamide for chlorpropamide in patients requiring and receiving maximal combined sulphonylurea and biguanide therapy, did not, however, improve diabetic control. As expected, glibenclamide was not

Preliminary Communications

of this

a

suitable alternative medication for those diabetics on insulin, who continued

satisfactorily maintained to require the hormone.

The mechanism of action of the new sulphonylurea presumed, as in the case of other sulphonylureas, to be through stimulation of insulin release from pancreatic islet cells.l The present evidence of increased fasting levels of insulin in healthy adults after glibenclamide and their altered response to oral glucose supports this view. There was, however, no direct relation between individual rises in plasmainsulin and the relative lowering of blood-glucose in healthy adults and, in the diabetic patients, fasting levels of plasma-insulin 3 hours after glibenclamide were similar to those recorded 24 hours after its administration. Whether this implies a qualitative as well as quantitive difference between glibenclamide and other sulphonylureas, 13,14 a potentiation of insulin action by the drug,! or greater tissue accessibility of the hormone under the influence of glibenclamide, is not known, however, and requires further study. It is also possible, even though no significant changes in plasma levels of G.H. were observed after glibenclamide, that changes in secretion-rate and half-life of G.H. might contribute to the hypoglycxmic effect of the drug, and we are now investigating this.

is

TOXICITY OF FOLIC ACID GIVEN IN PHARMACOLOGICAL DOSES TO HEALTHY VOLUNTEERS

JOANNA BARNES

RICHARD HUNTER Friern

Hospital,

London N.11

H. F. OAKELEY St. Thomas’s

Hospital,

London S.E.1

D. M. MATTHEWS Westminster Medical School, London S.W.1

elucidate the effect of folic pharmacological doses (15 mg. on daily) serum-vitamin-B12 levels in a group of fourteen healthy volunteers was abandoned after 1 month of a projected 3-month period because of the unexpected development of increasingly disturbing toxic effects in the majority. Most of the subjects experienced mental changes, sleep disturbance and gastrointestinal symptoms. Possible mechanisms of these effects are discussed. Sum ary

A trial acid in

to

INTRODUCTION

WE have reported a significant fall in serumvitamin-B12 levels after administration of folic acid (15 mg. daily) for 3 months to a group of long-stay patients on anticonvulsant medication in a psychiatric hospita1,1 To test whether this happened in healthy individuals we started a trial of folic acid in a group of fourteen volunteers. By the end of the first month most of them had symptoms which caused increasing discomfort and began to interfere with their work, and the experiment No such toxic effects had been was abandoned. " anticipated, so the trial was not done on a blind basis. VOLUNTEERS

AND

METHODS

Of the fourteen volunteers six were men and eight were women. Their ages ranged from 22 to 57 with a mean of 36 years. Serum-folate was estimated with Lactobacillus casei by the method of Waters and Mollin and serum-vitamin-B12

62

by a modification of the method of Hutner et al.3 using Euglena gracilis Z strain. Hxmoglobin, serum-folate, and vitamin-B12 levels were determined at the beginning and end ot the trial. Volunteers were given 3 months’ supply of 5 mg. tablets of folic acid and instructed to take one three times a day with meals. In the event of a dose being accidentally omitted, they were to take two tablets on the next occasion. As a precautionary measure to ensure that vitamin-B12 deficiency and possibly neuropathy were not allowed to develop, the volunteers were interviewed weekly by an independent observer who had been told to note any untoward symptoms. RESULTS

Table I shows the results of heamoglobin, serumfolate, and serum-vitamin-B12 estimations at the beginning and end of the trial (mean ±S.E.M. or TABLE I-HÆMATOLOGICAL FINDINGS AT BEGINNING AND END OF

1 MONTH

before retiring. Two dreamed in colour for the first time to their recollection. One had nightmares in the first week but slept normally thereafter. Eight complained of malaise and irritability which became more pronounced as the trial progressed. In five it was noted by relatives or friends before the volunteer himself became aware of the change. Five became overactive and excitable, and three of these felt a definite sense of increased energy and wellbeing. Two subsequently became depressed. Three had brief spells of mild confusion and impaired judgment and complained of increasing difficulty in

concentrating. After discontinuing the drug the majority improved rapidly: they regained normal sleep and dream patterns within a week: gastrointestinal symptoms subsided in the second week but normal mental function fully restored in all until the end of the third week. Three volunteers, however, initially became worse off medication and only began to lose their symptoms a week later. was not

DISCUSSION

There was no correlation at either point between incidence or severity of toxic effects and serum-folate or vitamin-B12 levels. One volunteer remained unaffected throughout. The remaining thirteen had a variety of symptoms which fell broadly into two groups-gastrointestinal and nervous (table II). Eight complained of gastrointestinal disturbances - anorexia, nausea, abdominal distension and discomfort, and flatulence. In four these developed in the first week (in two within 72 hours), and in the remainder in the third and fourth weeks. Six simultaneously reported a constant bad or bitter taste which impaired normal taste. Three women lost about half a stone (3 kg.) in weight. Nine experienced altered sleep patterns: two slept more deeply and dreamed less, one had difficulty waking in the morning, and six complained of varying degrees of insomnia either waking intermittently throughout the night or an hour or two before their usual time. This became increasingly distressing. Seven said they had vivid anxiety dreams. One reported aggravation after taking 10 mg. of folic acid

range).

TABLE II-TOXIC EFFECTS REPORTED BY VOLUNTEERS

Folic acid has been regarded chiefly as a vitamin concerned in hxmopoiesis and as non-toxic except in a few specific situations.’,’ A few examples of sensitivity reactions with skin rashes, malaise, and bronchospasm have been reported5,6; and folic acid given to patients on anticonvulsant medication may lead to escape from fit control or even status epilepticus.7,8 The best established and most serious danger of giving folic acid is that in patients with pernicious anaemia it may precipitate neurological lesions either by mobilising depleted vitamin-B12 stores or by direct neurotoxic action,9,10 but this is said to happen only after 3 months of folate therapy.lO Our volunteers’ symptoms were not related to changes in haemoglobin or blood-films, nor apparently to fluctuations in serum-vitamin-BI2 levels caused by folic acid since at the end of the trial no clear or general trend had emerged. The rapidity of onset of toxic symptoms in some and their uniform pattern, whether they cases developed early or late in the trial, suggested that folic acid might have a specific effect on cerebral metabolism, either by passing through the blood/ brain barrier or by concentrating in cerebrospinal fluid (c.s.F.). Under normal conditions the level of folic acid in c.s.F. is considerably higher than in serum, and may be as much as 5-10 times as high.11-13 If c.s.F. folate levels are correlated with serum-folate, then very high levels of c.s.F. folate are likely to have been reached in our volunteers because their mean serum-folate levels rose from 4-5 ng. per ml. at the beginning of the trial to more than 120 ng. per ml. at the end. Folate derivatives can influence brainamine synthesis in vitro,14-16 so it could be that the symptoms of folic-acid intoxication we observed may be related to brain-amine change. If this hypothesis can be substantiated, it may also account for the "alerting" effect. of folic acid when given to patients on anticonvulsant medication observed occasionally by ourselves and remarked on by Reynolds.17 This has been attributed to folic acid reversing the "mental" effects of anticonvulsants,17

63

but may have been caused by a direct stimulating effect of folic acid comparable to that observed in This effect may also be our healthy volunteers. for the increased feeling of wellbeing responsible in patients receiving deep X-ray treatment reported when they are given folic acid.l Whether the gastrointestinal disturbances are mediated through a similar central pathway or resulted from a direct effect on the gut remains conjectural. But it is of interest that folic acid seems to be concentrated in saliva,l9 a fact which may explain the bad or bitter taste complained of by some of our volunteers. We thank Dr. Gerald Curzon, of the Institute of Neurology, for discussing with us the possible role of folic acid in cerebralamine metabolism; Miss Catherine Duncan, of the M.R.C. Statistical Research and Services Unit, for advice on statistical aspects; and Mr. N. Jollyon Smith for help in gathering data. Requests for reprints should be addressed to R. H. REFERENCES 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Hunter, R., Barnes, J., Matthews, D. M. Lancet, 1969, ii, 666. Waters, A. H., Mollin, D. L. J. clin. Path. 1961, 14, 335. Hutner, S. H., Bach, M. K., Ross, G. I. M. J. Protozool. 1956, 3, 101. Goodman, L. S., Gilman, A. The Pharmacological Basis of Therapeutics; p. 1435. New York, 1966. Chanarin, I. The Megaloblastic Anæmias; p. 958. Oxford, 1969. Woodliff, H. J., Davis, R. E. Med. J. Aust. 1966, i, 351. Chanarin, I., Laidlaw, J., Loughridge, L. W., Mollin, D. L. Br. med. J. 1960, i, 1099. Reynolds, E. H. Lancet, 1967, i, 1086. Wintrobe, M. M. Clinical Hæmatology; p. 485. London, 1967. Chanarin, I. The Megaloblastic Anæmias; p. 588. Oxford, 1969. Kallström, B., Nylof, R. Acta psychiat. scand. 1969, 45, 137. Wells, D. G., Casey, H. J. Br. med. J. 1967, iii, 834. Herbert, V., Zalusky, R. Fedn Proc. 1961, 20, 453. Nagatsu, T., Levitt, M., Udenfriend, S. J. biol. Chem. 1964, 239, 2910.

Kaufman, S. in The Enzymes (edited by P. D. Boyer, H. E. Lardy, and K. Myrback); vol. VIII, p. 373. New York, 1963. 16. Gal, E. M., Armstrong, J. C., Ginsberg, B. J. Neurochem. 1966, 13, 643. 17. Reynolds, E. H. Brain, 1968, 91, 197. 18. Doan, C. A. Am. J. med. Sci. 1946, 212, 257. 19. Markkanen, T., Makila, E. Lancet, 1965, i, 1118. 15.

air and that in the blood of

exposed individuals or in man, lead can that, suggesting populations accumulate as a result of increasing environmental 8

contamination.

Any effect of the absorbed lead

health will depend on the degree of accumulation. The generally accepted " safe " upper limit for the concentration of lead in the blood of persons exposed industrially is some 70-80 g. per 100 ml.,l2 and mean values for on

occupationally unexposed populations

are

usually

between 15 and 25 ug. per 100 ml.3.7-io These levels are considered by most workers to be without any biological effects. Inhibition of hxm synthesis is one manifestation of lead toxicity; and measurements of intermediate products of this metabolic chain-e.g., coproporphyrin and 8-aminolsevulinic acid (A.L.A.) in urine-have long been used as quantitative tests of the response of the organism to surplus lead 3 " and have thus formed one basis for the evaluation of what is harmful and what is not. The finding that erythrocyte A.L.A. dehydrogenase activity is specifically inhibited by lead has provided a new, specific, and sensitive method for the detection of early lead effect.11-13 We have used this test to see if measurable biological effects due to present levels of urban exposure to lead can be found in man. MATERIAL

AND

METHODS

Blood-lead levels and erythrocyte A.L.A. dehydrogenase activity were measured in 26 healthy medical students (16 males, 10 females) with no present or previous occupational exposure to the metal. The concentration of lead in whole blood was measured after wet ashing using a dithizone method. 14 The standard deviation of duplicate measurements was ±1-3 g. per 100 ml. A blind control of the lead estimation gave the following result (the concentration in the blank sample recorded as 9 .g. per 100 ml.) :e

ENZYME INHIBITION BY LEAD UNDER NORMAL URBAN CONDITIONS

S. HERNBERG Institute

J. NIKKANEN

of Occupational Health, Haartmaninkatu 1, Helsinki 29, Finland

A close negative correlation was found between the concentration of lead in blood and the activity of erythrocyte &dgr;-aminolævulinic acid dehydrogenase in 26 healthy individuals, never exposed occupationally to lead. The results indicate that present levels of environmental contamination with lead can produce a measurable biochemical alteration Sum ary

dehydrogenase was measured from fresh heparinised whole blood according to Bonsignore’s method.12 The results were corrected for hxmatocrit value, and the enzymatic activity was calculated from the formula: 1000 x 12-5 (A6o-Ao)fhaematocrit (%) A60 and Ao are the absorbencies at 60 and 0 minutes, respectively, and 12-5 is the dilution correction. A.L.A.

in man. INTRODUCTION

LEAD is absorbed into the human organism by and digestive routes and is distributed to organs and tissues by the bloodstream.’- Metabolism is complex-some lead being excreted and some deposited; the rest forms a metabolically active pool reflected by the blood-level of lead. 1,2 As a result of growing industrial use and the introduction of alkyl lead compounds as petrol additives, exposure to lead is substantially above 3-9 the background of absorption from food and drinking-water. Epidemiological and experimental data point to a logarithmic correlation between the concentration of lead in the

respiratory

Relation between blood-lead and activity of erythrocyte A.L.A. dehydrogenase of 26 persons never occupationally exposed to lead.