781 INHIBITION OF PLATELET AGGREGATION AND THROMBOXANE SYNTHESIS BY ONION AND GARLIC SIR,-Platelet aggregation is reported to be inhibited by both onion (Allium cepa)1,2 and garlic (A sativum).’ Work on cyclic endoperoxides4 and thromboxane A2 has revealed the role of arachidonic-acid metabolites in platelet function. These short-lived compounds induce platelet release and aggregation reactions. Non-steroidal, anti-flammatory drugs such as aspirin and indomethacin block conversion of arachidonic acid to thromboxane by inhibiting cyclo-oxygenase, and this accounts for the anti-platelet activity of these drugs. Arachidonic acid is also converted by a platelet lipoxygenase.6 to a monohydroxy fatty acid (H.E.T.E.).’ Very little is known about the biological functions of this alternate path-

way.8 1. Phillips, C., Poyser, N. L. Lancet, 1978, i, 1051. 2. Baghurst, K. I., Raj, M. J., Truswell, A. S. ibid. 1977, i, 101. 3. Bordia, A. Atherosclerosis, 1978, 30, 355. 4. Hamberg, M., Samuelsson, B. Proc. natn. Acad. Sci. U.S.A. 1974, 71, 3400. 5. Hamberg, M., Svensson, J., Samuelsson, B. ibid. 1975, 72, 2994. 6. Nugteren, D. H. Biochim. biophys. Acta, 1975, 380, 299. 7 Hamberg, M., Samuelsson, B. Proc. natn. Acad. Sci. U.S.A. 1974, 71, 3400. 8. Goetzl, E. J., Woods, J. M., Gorman, R. R. J. clin. Invest. 1977, 59, 179. 9. Bailey, J. M., Bryant, R. W., Feinmark, S. J., Makheja, A. N. Prostaglan-

dins, 1977, 13, 479.

We have isolated anti-platelet activity as an oily chloroform of onion which was further purified by chromatograThe phy. inhibitory activity was associated with a heat-stable, non-polar material not inactivated by mild acid and alkali. Similar anti-platelet inhibitory properties were observed with both onion and garlic oil (the median inhibiting concentration per ml platelet-rich plasma was 30-100 ug for different samples of human and rabbit platelets). In addition, platelets incubated with onion or garlic inhibitors and (1-14C)- arachidonic acid showed striking changes in the pattern of metabolites formed in comparism with controls or platelets incubated with the cyclo-oxygenase inhibitor indomethacin (see figure). Most apparent was the almost complete suppression of thromboxane B2 synthesis, the partial inhibition of H.E.T.E. synthesis, and the appearance of a new metabolite in the region of the hydroxy fatty acids (H.H.T. and H.E.T.E.). This new metabolite has been identified as a product of the platelet lipoxygenase reacextract

tion.

Oxygen consumption measurements confirm that these materials inhibit the platelet cyclo-oxygenase since both the rates and total amounts of oxygen consumption fell. Similar inhibition of sheep vesicular gland cyclo-oxygenase was obtained with onion oil whereas garlic oil was less effective. Gas chromatographic and mass-spectro-metric analyses of active extracts of onion and garlic show differences in several major components which may relate to the observed differences in biological activity. Our results thus indicate that both members of the Allium family, commonly used in the diet, contain chemically similar compounds which inhibit platelet aggregation by blocking thromboxane synthesis. Department of Biochemistry, George Washington University School of Medicine,

Washington, D.C. 20037, U.S.A.

AMAR N. MAKHEJA JACK Y. VANDERHOEK J. MARTYN BAILEY

UNITS FOR IMMUNOREACTIVE TRYPSIN

SIR,—The radioimmunoassay for human immunoreactive

trypsin (I.R.T.)

described in 1977’ and

now

commercially avail-

(’RIA-gnost Trypsin’, Behringwerke, Marburg) is being increasingly, as in the paper by Dr Crossleyand her colleagues (March 3, p. 472). There may be some confusion about the units for I.R.T. concentrations in biological fluids. Elias et al.1 expressed results as µg trypsin standard/I, referring to a semipurified trypsin preparation Ag5. We have worked out fac-

able used

allow conversion of Ag5 to mass of pure enzyme and have derived the hypothetical concentration of enzymic

tors to

activity.2 Highly purified

DISTANCE FROM ORIGIN

15 CM

Differential inhibition of 14C-arachidonate metabolism in platelets by onion oil and by indomethacin. The figure shows the chromatographic separation and assay of the platelet products from (1-14C)-arachidonic acid (AA). 1 ml of washed and resuspended platelet-rich plasma was preincubated with 0.5 ul of ethanol without inhibitor (A) or containing 42 ug of onion fraction or 10 µg of mdomethacin (C) for 2 min at 37°C before the addition of 1-"C;-AA. The reactions were stopped by acidification with 10% formic acid followed by ethyl-acetate extraction and thin-layer chromatography in C, solvent system.9 The quantitative distribution of radioacwas determined by use of a one-dimensional TLC Vanguard 93 autoscanner.

human trypsin, prepared by a modification of the method of Temler and Kägi,3 was used to estimate both the enzymic and the immunological "purity" of Ag5. The enzymic activity of the pure material was assessed at 119 katal/kg.4 The semipurified enzyme Ag5 had an activity of 40 katal/kg before inhibition for use as standard in the test kit. Immunologically, the semipurified trypsin had 45% of the potency observed with the highly purified enzyme, the latter being used both as tracer and immunogen in the radioimmunoassay. concentrations measured in this radioimmunoassay as ug pure trypsin/1 by calculating 45", of the value or in molar terms by converting the assayed assayed value to pure trypsin concentration and then dividing this value by its molecular weight (23 000). The mean normal serum-i.R.T. value of 272 µg Ag5/1 given by Elias et al.’ thus conI.R.T.

can

be

given

1. Elias, E., Redshaw, M, Wood, T. Lancet, 1977, ii, 66. 2. IUPAC-IUB 2nd Report of Commission on Enzymes; p. 26. 1973. 3. Temler, R S., Kägi, J. H. R. Enzyme, 1977, 22, 249. 4. Schwert, G. M., Takenaka. Y. Biochim. biophys Acta, 1955, 16,

Amsterdam,

570.

782 verts to

pure

122 µg pure immunoreactive

trypsin/1 or

5-3nmol

I.R.T./l serum.

If the biological (enzymic) activity is considered (in the absence of an international reference preparation for human trypsin) values in ;j!.g/I could be equated with enzymic activity units of Behring Institute pure trypsin/1 (µkatal/l). Since with all proteins, especially enzymes, the definition of "purity" is open to a wide variety of interpretations and since serumtrypsin is assayed bound to inhibitors, this figure is somewhat hypothetical. Nevertheless, the mean normal serum-I.R.T. value of 272 ug Ag5/1 could be given as 14.5 µkatal pure trypsin enzymic activity/I serum. These aspects should be taken into account when considering the clinical implications of published trypsin values5.6 and when comparing results from different centres. Hoechst UK Ltd, Walton Manor, Walton, Milton Keynes,

Buckinghamshire MK7 7AJ

T. P. WOOD

M. R. REDSHAW R. H. ROUSELI

DIALYSIS DEMENTIA, ALUMINIUM, AND TETRAHYDROBIOPTERIN METABOLISM

SIR,—Mr Leeming and Professor Blair (March 10, p. 556) suggest that dihydropteridine reductase (D.H.P.R.) inhibition may be a cause of aluminium neurotoxicity and hence of dialysis dementia. If aluminium is a major factor in the development of this disorder, there may be another metabolic explanation for the mechanism. Patients on chronic hæmodialysis have an excess of aluminium not only in the brain but also in other organs (including bone), the excess correlating with the duration of hoemodialysis. In particular, there is excess aluminium in the grey-matter. Synaptosomes, which are found only in grey-matter, store neurotransmitters, these are taken up for storage by an energy-requiring process involving A.T.P. Metal chelates are important in the storage and transport of neurotransmitters (and this may be clinically relevant in the development of parkinsonism in manganese toxicity, as well as in the trace-metal disturbances found in Huntington’s chorea). The formation of ternary complexes (A.T.P.-metal-noradrenaline) is important in catecholamine binding.2 Such complexes would incorporate free noradrenaline. Aluminium may play a part in this mechanism by forming ternary complexes3 thus reducing the free total noradrenaline available to the synaptosomes. One can only speculate as to the role of aluminium in forming these complexes with other neurotransmitter substrates. Catecholamine binding in conduction with the known loss of amines into the dialysate4 may explain the fluctuation of symptoms in relation to periods of dialysis and, in time, the gradual loss of this feature. Sites of catecholamine storage are all within grey-matter, the distribution being similiar to that of aluminium in encephalopathic brains. Any hypothesis incorporating a role for aluminium in the production of dialysis dementia must take into account the distribution of aluminium, and, in the context of Leeming and Blair’s proposition, the histochemical distribution of D.H.P.R. is obviously important. Further work should attempt to link aluminium deposition with known relative concentrations of stored neuroamines, and ternary complexes should be estimated in subcellular fractions from fresh biopsy or necropsy material.

DIETARY FIBRE AND DIABETES

SIR,—We are glad that Dr Williams and Dr James (March 17, p. 612) reaffirm our comment that, to be effective, fibre should be intimately mixed with the major carbohydrate portion of the food, though not necessarily all the carbohydrate portions. This does not mean that it must be cooked with the food, as illustrated by the effect of guar added to soup and consumed together with bread.’ Nevertheless the effect is best in the high-carbohydrate meals where guar has been added, as shown by the work of Dr Goulder (March 17, p. 612). Though we agree with the intuitive statement of Williams and James that long-term trials are necessary we do not feel that this follows from their published work showing that guar unmixed with the carbohydrate part of the meal is ineffective. Nor have they grounds at present for suggesting that "natural foods" may be more effective and less toxic as there is virtually no comparative data on this subject. On the contrary, wheat bran and high-fibre wheat products may reduce serum calcium2 and iron3 and in Iran are implicated in zinc deficiency with dwarfism, anaemia, cirrhosis, and hypogonadism,4 but at present no data exist on guar or other refined fibres in longterm use. However, it appears that there is a natural and a pharmacological aspect of fibre5 and both may have their place in therapy. We are disappointed in the stand which Williams and James take on sugar in the diabetic diet. As pointed out by Jenkins5 this would tend to reduce the action of any fibre. The successful development of a glycosidehydrolase inhibitor for the treatment of diabetes has been aimed specifically at reducing the effects of dietary sucrose on blood-sugar.6-8 Furthermore starches produce flatter blood-glucose responses than do sugars.9 Finally the use of low-starch/high-sugar foods is contrary to the fibre hypothesis.’" Dr Dewar and her colleagues (March 17, p. 612) report results on six subjects who were nauseated by guar and on whom urinary xylose-excretion measurements at 5 h revealed no difference from controls. The choice of a 5 h absorption test was unfortunate since previous data," cited by Dewar et al., show that in the first 2 h after ingestion there is a significant drop in xylose recovery with guar, while at 4-6 and 6-8 h a significant increase was seen such that over 8 h there was no significant difference. This indicated that there was a delay in absorption with no evidence of malabsorption. Thus at 5h the delayed xylose is continuing to appear and, with individual variation between subjects, the total xylose recovery in 5 h will certainly be misleading. In addition the presence of nausea in the subjects studied makes the interpretation of transit, absorption, and intestinal motility very difficult. Dewar et al. comment on the impossibility of using guar due to unpalatability, and they lament the time wasted by so many in unsuccessful attempts and the fact that investigators were not warned of this problem. We have successfully mixed guar with orange juice and soup and made bread with it,"-" and, 1.

Wolever, T. M. S., Taylor, R. H., Goff, D. V., Ahern, J. Lancet, 1979, i,

435. 2. Heaton, K. W., Pomare, E. W. ibid. 1974, i, 49. 3. Jenkins, D. J. A., Hill, M. J., Cummings, J. H. Am J. clin. Nutr. 1975, 28, 1408. 4. Reinhold, J. G., Faradji, B., Abaidi, P., Ismail-Beigi, F. J. Nutr. 1976, 106, 493. 5. Jenkins, D. J. A., Nineham, R., Craddock, C., Craig-McFeely, P., Donald-

J. Lancet, 1979, i, 434. J., Sherif, I. T., Noy, G. A., Alberti, K. G. M. M. Br. med. J. 1979, i, 220. 7. Taylor, R. H., Jenkins, D. J. A., Nineham, R. Gut, 1978, 19, 969. 8. Taylor, R. H, Jenkins, D. J. A., Nineham, R., Sarson, D., Bloom, S. R, Alberti, K. G. M. M. Gastroenterology (in the press). 9. Crapo, P. A., Reaven, G., Olefsky, J. Diabetes, 1977, 26, 1178. 10. Trowell, H. C. in Refined Carbohydrate Foods and Disease (edited by D. P Burkitt and H. C. Trowell); p. 246, London, 1978. 11. Jenkins, D. J. A., Wolever, T. M. S., Leeds, A. R., Gassul, M. A., Haisman, P., Dilawari, J., Goff, D. V., Metz, G. L., Alberti, K. G. M. M. Br. med J 1978, i, 1392. 12. Jenkins, D. J. A., Wolever, T. M. S., Haworth, R., Leeds, A. R., Hockaday, T. D. R. Lancet, 1976, ii, 1086. son, K., Leigh, T., Snook,

6. Walton, R.

Department of Neurology, Institute of Neurological Sciences, Glasgow GS1 4TF

IAN BONE

MYFANWY THOMAS

5 Adrian, T.E, and others Clin. Sci mol. Med 1978, 54, 13P. 6. Dandona, P., Elias, E., Beckett, A G. Br. med.J 1978, ii, 1125. 1. Rajan, K. S., Davis, J. M, Colburn, R. W. J. Neurochem. 1971, 18, 345. 2 Colburn, R W., Maas, J W. Nature, 208, 37 3 Kirshner, N. J. biol. Chem 1962, 237, 2311. 4. Wooton, I. D. P in Scientific Basis of Medicine: annual review; p. 234. Lon-

don, 1963.