Clina’ca Chimica Acta, 68 (1976) 141-146 @ Elsevier Scientific Pubfishing Company, Amsterdam - Printed in The Netherlands

CCA 7654

TOXIC~L~G~UFMET~YLM~T~AC~Y~ATE~T~E~ATE~F DI~APP~A~A~CE OF METHYL MET~ACRYLATE IN VITRO

IN ~~~~A~

BLOOD

&A. CORKILL &,E.J. LLOYD axP. HOYLE *, D.W.G. CROUT a*, R.S.M LXNC b, ML. JAMES b and R.J. PIPER c

(Received November 10, 1975)

1. The rate of disappe~a~~e of methyl methac~lat~ in blood has been determined using an isotope dilution technique. disappears 2. At a concentration of lo_4 mol dmS3, methyl methac~late with pseudo first order kin~ti~s~ 3. The half-life of methyl metba~~ylate in blood at 37°C lies in tine range 20-40 min. 4. The half-life showed no dependence on the age or sex of the blood donor. 5. A major, possibly the only, pathway of metabalism is by hydrolysis to methacrylie acid,

Introduction Polymethylmethac~late is widely used as a cement in orthopaedi~ surgery [l-43 . Tke cement is prepared from a mixture of ~repolyme~sed material which contains a small amount of an initiator such as be~2oylper~~ide and liquid monomers usually stabilised with hydroquinone and containing an accelerator such as NJ&dimethyl-@oluidine. During surgical procedures in which the cement is used, notably total hip replacement, a certain amount of mon.omer enters the bloodstream, Hypotensive effects noted during surgery have been attributed to the ~~diovascul~ action of circulating monomer [3,5--9t and it was therefore considered desirable to examine the rate of entry of methyl methacrylate into the bloodstream during surgery and, iudepende~tly, to efucidate its pathway of metabolism. * To

whom reprint requests should be addressed.

142

Because of its precision, the isotope dilution method was chosen for analysis of methyl methacrylate in blood in the 10m5- 10e4 mol dme3 (l-10 pg Crne3) range. It was also expected to be adaptable to the analysis of methyl methacrylate in vivo using the reverse double isotope derivative method, a modification which would avoid the highly undesirable practice of exposing patients to 14C-labelled monomer as in certain reported investigations of methyl methacrylate transport in vivo [ 5,101. Materials and methods Chemicals [ 14C]Methyl methacrylate (472 /.Ki g-l) was purchased from the Radiochemical Centre, Amersham, Bucks, England. Inactive methyl methacrylate, stabilised with 0.1% hydroquinone, was purchased from B.D.H., Ltd. (Poole, England). N-Phenyl-C-benzoylnitrone (II) was prepared by the method of Huisgen et al. [ll] . On reaction with methyl methacrylate (I) it gave the adduct (III) (methyl 2-phenyl-3-benzoyl-5-methylisoxazoline-5-carboxylate) as yellow needles m.p. 143.5-144.5”C (Lit. [ll] 140-141.5”C). 2-Phenyl-3-benzoyl-5-methylisoxazoline-5-carboxylic acid (IV) was prepared by adding methacrylic acid (5.2 cm3, 5 mmol) to a solution of N-phenyl-C-benzoylnitrone (1.13 g, 5 mmol) in ethanol (20 cm3). The solution was heated at 50°C for 20 min and cooled to give the adduct (IV) as yellow needles (1.4 g) m.p. 131-132°C. (Found: C, 69.85; H, 5.50; N, 4.47%. ClsH17N04 requires C, 69.45; H, 5.47; N, 4.50%). Blood samples Two samples (Nos. 7 and 8, Table I) were obtained from patients at the Princess Elizabeth Orthopaedic Hospital, Exeter. The remaining samples were obtained from healthy volunteers. After collection, the samples were placed in heparinized tubes and used for the metabolic studies within two hours. Determination of methyl methacrylate in blood The blood or serum sample (10 cm3), incubated at 37”C, was treated with a solution of [14C]methyl methacrylate (100 pg) in water (0.1 cm3). At timed intervals, aliquots (1 cm3) were removed and transferred to a tube containing inactive methyl methacrylate (50 mg) in halothane (2 cm3). The mixture was shaken mechanically for 1 min, centrifuged, and a portion (1.75 cm3) of the halothane layer was treated with excess (250 mg) nitrone (II). The mixture was heated to 50°C for 45 min and cooled to precipitate the adduct (III) which was recrystallised from ethanol. It was found, by repeating the extraction of the blood, that this one-step procedure resulted in the removal of all halothane-extractable methyl methacrylate from the sample. It was also found that one recrystallisation of the adduct from ethanol was sufficient to give material of constant specific activity. The rate of hydrolysis of methyl methacrylate in water was determined using the above procedure. Radioactivity determination The radioactivity of the labelled adduct was determined in dioxan-based scintillation fluid (B.D.H., Ltd., Poole, England) using a Packard 2000 Series

143

Liquid Scintillation Counter. For counting, lo-mg samples were taken in duplicate. Sufficient counts were accumulated to give a statistical error of 40% conversion into methacrylie acid after 90 min represents a major if not the sole, initial step in the metabolism of methyl methacrylate. Further evidence of the non-trivial hydrolysis of methyl methacrylate was obtained by incubating the ester in deionised water at 37°C. The half-life under these conditions was found to be >3.5 hours (three determinations). Discussion Observation of the enzymatic hydrolysis of methyl methacrylate is significant in relation to reported studies of methacrylate transport in patients undergoing hip replacement, in which methacrylate concentrations were determined by simple radioactivity determinations [ 5,lO 3 . The concentrations reported were of the same order of magnitude as those used in the present studies. However, the ester used in these experiments was labelled in the O-methyl group. Hydrolysis would therefore lead to the production of radioactive methanol and non-radioactive methacrylic acid. The possible presence of significant undetected quantities of methacrylic acid in the circulatory system was therefore not taken into account. Since methacrylic acid exerts a significant cardiovascular effect on the isolated, perfused rabbit heart [ 121, its formation by enzymatic hydrolysis of the ester must be considered in relation to the overall cardiovascular effect of methyl methacrylate in vivo. The hydrolysis of methyl methacrylate to methanol and methacrylic acid is of interest in relation to reported inconsistencies [lo] in the cbncentrations of methyl methacryiate in the circulation of patients undergoing hip surgery as

145

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Fig.

1.

determined by radioactivity measurements and gas chromatography. The apparent methyl methacrylate concentrations, as determined by radioactivity measurements, were higher than those measured by gas chromatography and reached a maximum only after the gas chromatographically-determined concentrations had already reached a maximum and begun to decline. The authors [lo] suggested that the levels determined by radioactivity measurements were distorted owing to the presence of labelled oligomers and metabolites of methyl methacrylate. Our results suggest that a significant proportion of the radioactivity observed was due to labelled methanol produced by hydrolysis of the (Omethyl)-labelled methyl methacrylate. Since the location of the label in commercially available [3H] - and [ 14C] methyl methacrylate made it unsuitable for use in following the production and further metabolism of methacrylic acid, a synthesis of [ 3-3H] methyl methacrylate ([ 3-3H] methyl 2-methylpropenoate) was devised. This material was used for certain of the determinations of the rate of methyl methacrylate disappearance. (Experiments 3 and 4, Table I). Further studies of methacrylic acid metabolism using this labelled ester are in progress.

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I

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146

The production of this acid is significant in that, as the coenzyme A ester, it is an intermediate in the catabolism of valine [13], being converted ultimately into methylmalonyl-CoA, succinyl-CoA and P-aminoisobutyric acid (Fig. 1). It is therefore probable that if a methacrylic acid-coenzyme A ligase is present in the blood or other organs, this compound will be metabolised via a normal catabolic pathway, to be excreted as the metabolites noted above. Experiments are in progress to examine this possibility. It has been suggested by PantSek [14] that methyl methacrylate is metabolised according to the sequence shown in Fig. 2. This pathway was proposed on the basis of studies with rat liver slices in which malonate was shown to have no effect on the rate of methyl methacrylate metabolism, thus ruling out the participation of the citric acid cycle which would be necessary if methacrylate were to be metabolised to CO, via succinyl CoA, as in the postulated pathway given in Fig. 1. On the other hand, methyl methacrylate metabolism was strongly‘ inhibited by arsenite, which was taken as evidence for an oxidative decarboxylation step in the pathway. Since arsenite inhibits the citric acid cycle, the argument for the pathway shown in Fig. 2 rests on the observation that methyl methacrylate metabolism was not inhibited by malonate. These studies, however, only demonstrated the disappearance of methyl methacrylate, they did not demonstrate the metabolism of methyl methacrylate through to carbon dioxide. It is possible therefore that malonate inhibition might have resulted simply in the accumulation of methylmalonyl-CoA and succinyl-CoA. Since methylmalonic acid is a normal catabolite of valine in man it is clear that metabolism through to carbon dioxide is certainly not obligatory for all of the methacryl-CoA produced during valine catabolism. It cannot be concluded therefore that the catabolic pathway of Fig. 2 is established. Further investigations are in hand to resolve this question. Acknowledgements This investigation was supported by the National Crippling Diseases and by Howmedica International Division, 49 Grayling Road, London, N16 OBP, U.K.

Fund for Research into Ltd., North Hill Plastics

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Charnley, J. (1964) J. Bone Joint Surg. 46B. 518 Charnley. J. (1965) J. Bone Joint Surg. 47B. 354 Charnley, J. (1970) Acrylic Cement in Orthopaedic Surgery. Williams and Wilkins. Baltimore Holland, C.J., Kim, K.C., Malik, M.I. and Ritter, M.A. (1973) Clin. Orthop. 90. 262 Homsy, C.A., TulIos. H.S., Anderson, M.S., Differante, N.M. and King. J.W. (1972) CIin. Orthop. 83. 317 Kim. K.C. and Ritter, M.A. (1972) Clin. Orthop. 88,154 Phillips, H.. Cole, P.V. and Lettin, A.W.F. (1971) Br. Med. J. 3, 460 Thomas, T.A., Sutherland, I.C. and Waterhouse, T.D. (1971) Anaesthesia 26, 298 Modig, J., Olerud, S. and Malmberg, P. (1973) Acta Anaesth. Stand. 17, 276 Eggert. A., Huland, H., Ruhnke, J. and Seidl. H. (1974) Chirurgia 45, 236 Huisgen, R., Hawk, H.. Seidl. H. and Burger. M. (1969) Chem. Ber. 102, 1117 Mir, G.N., Lawrence. W.H. and Autian. J. (1973) J. Pharm. Sci. 62. 778 Rodwell, V.R. (1969) Metabolic Pathways, Vol. III (Greenberg, D.M., ed.), p. 198, Academic Press, New York and London PanttiZek, M. (1969) FEBS L&t. 2. 206