As part of an attempt to elucidate the mechanism of carcinogenesis by αβ-unsaturated y-lactones, a systematic study of the metabolism of a group of saturated and  αβ-unsaturated lactones was performed. It was shown that the saturated y-lactones are much more rapidly enzymatically hydrolysed than their αβ-unsaturated analogs. while the latter compounds are also conjugated with glutathione which could eventually lead to the excretion of mercapturic acids. On the whole, however. the saturated lactones are more rapidly metabolized

de Kock, D.H.

Raubenheimer, H.G.

English

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570LCM4S.A. MEDICAL JOURNAL(Supplement-Sou rh African Journal of Laboratory and Clinical Medicine)29 May 1971the following is observed: the predicted normal meanvital capacity for this man would be 3·45 litres accordingto 'Bantu values',' and 4·7 litres according to 'Whitevalues'." There is thus a predicted difference of 1·25 litres.If stem or sitting height of 87 cm is first used to cal-culate standing height, and if vital capacity is then pre-dicted from standing height, the following values will bederived: the Bantu male would be 171 cm tall, and wouldhave a vital capacity of 3·6 litres, and the White malewould be 167 cm tall and would have a vital capacity of4·7 litres. Thus the difference in vital capacity is onlyreduced to 1'1 litres.It may, therefore, be concluded that anthropomorphicdifferences in this example account for only a small pro-portion of the observed difference in vital capacity. Differ-ences of the same order can be found for other divisionsof total lung capacity. It is interesting to note that thisdisproportion is much more marked if the bodily pro-portions of Gilson's Welsh miners are substituted for thoseof the South African Whites.We wish to thank Dr N. F. Laubscher of the NationalResearch Institute for Mathematical Sciences, CSIR, for thestatistical evaluations: and Dr G. K. Sluis-Cremer, Directorof the Miners' Medical Bureau, Johannesburg, for co-operation.REFERENCESI. Smiley, W. G. and Augentine, D. L. (1927): J. Amer. Med. Assoc.,2. ~~ti~<;;~' Center for Health Statistics (1960 - 62): US Weight, Heigluand Selected Body Dimensions of Adults, series 11, No. 8. \Vashington,DC: US Government Printing Office.3. Aslell, E. A., D'Arcy-Hart, P. and McMichael, J. (1939): Proc. Roy.Soc. Med., 5B, 502.4. Gilson, J. C., Hugh-Jones, P., OIdham, P. O. and Meader, F. (1955):Spec. Rep. Ser. Med. Res. Coun. (Lond.), No. 290.5. Johannsen, Z. M. and Erasmus, L. D. (1968): Amer. Rev. Resp. Dis.,97, 585.6. Cotes, J. E. (1965): Lung Functions. Oxford: Blackwell ScientificPublications.7. Flemming, P. W., Ward, 1. S., Wyndham, C. H. and Joffe, A. (1964):S. Afr. J. Med. Sei., 29, 27.8. Kory, R. c., Callahan, R., Boren, H. G. and Syner, J. C. (1961):Amer. J. Med., 30, 243.METABOLISM OF GAMMA·LACTONES BY RAT LIVER PREPARATIONS IN VITRO*D. H. DE KOCK AND H. G. RAUBENHElMER, Department of Chemistry, University of StellenboschSUMMARYAs part of an attempt to elucidate the mechanism ofcarcinogenesis by af3-unsaturated y-lactones, a systematicstudy of the metabolism of a group of saturated and af3-unsaTUrated lactones was performed. It was shown thatthe saturated y-lactones are much more rapidly enzymati-cally hydrolysed than their af3-unsaturated analogs. whilethe latter compounds are also conjugated with glutathionewhich could eventually lead to the excretion of mercap-turic acids. On the whole, however. the saturated lactonesare more rapidly metabolized.Among the series of y-Iactones, marked biological activi-ties are often associated with the presence of unsaturationin the lactone ring.' Apart from f3-propiolactone, a com-pound with a highly strained ring structure, and aflatoxin,a complicated a-lactone, some y-lactones possess carcino-genic activity.'-' It appeared that af3-unsaturation of they-Iactones is also desirable for this activity. In the presentwork a systematic study of the metabolism of a groupof saturated and af3-unsaturated y-Iactones (Fig. 1) byR CH-Z-CH2 R..... rH=lH,I I 'c ~o/'C ......... /C=O R'/' """"'0/R' 01. R = R' = H IV. R R' = Hll. R = H; R' = CH.' V. R H; R' = ClLIll. R = R' = CH. VI. R R' = CH.Fig. 1. y-Iactones employed in this study: I. y-butyrolac-tone; ll. y-valerolactone; Ill. y-isocaprolactone; IV.y-crotonolactone; V. 4-hydroxypent-2-enoic acid y-Iactone(,a-angelica lactone); VI. 4-hydroxy-2-enoic acid y-Iactone.rat liver preparations was undertaken. Metabolic reac-tions which were studied were hydrolysis of the !actones,formation of conjugates with glutathione which is theinitial reaction leading to the formation of mercapturicacids: and hydroxylation of the unsaturated lactones."Date received: 15 October 1970.MATERIAL AND METHODSLactones I, II and V were obtained commercially whilelactones Ill, IV and VI were synthesized as describedby Haynes and Jones.'LaCTOne Hydrolysis AssayA rat liver homogenate (10% wJv) in 0'15M tris-histidine buffer (pH 7'4) was centrifuged at 10000 G toremove mitochondria and nuclei which contained no lac-tone-hydrolysing activity. Of the supernatant 1'0 mI wasadd~d to 1·0 mIlD mM lactone solution in de-ionizedwater and the mixture incubated at 3rC for the appro-priate time. Thereupon the concentration of the unhydro-lysed lactone was determined by the reaction with alkalinehydroxylamine followed by colorimetry in acid ferricchloride." For this purpose 1·0 ml 2M hydroxylaminewas added to each incubation mixture followed by 0'1ml 3N sodium hydroxide and 0·6 ml of a reag~nt con-sisting of a 1: 1 mixture of 96% ethanol and 10% ferricchloride in 4N hydrochloric acid. The proteins wereprecipitated by addition of 1·0 ml 0·1 M trichloroaceticacid to the mixtures and subsequent centrifugation. Finallythe optical density of the purple supernatant was mea-sured at 510 nm. Control determinations in which theliver preparation was boiled before incubation were alsocarried out.Reaction with GlutathioneA rat liver homogenate (10% wJv) in 0'05M phosphatebuffer (pH 7'4) was centrifuged at 700 G for 10 minutesto remove nuclei and unbroken cells followed by centri-fugation of the resulting supernatant at 120000 G for40 minutes, Of this supernatant samples of 2·5 mI wereadded to' a mixture of 2·5 ml 10 mM lactone solution inwater and 5·0 ml 5 mM glutathione in 0'05M phosphatebuffer (pH 7'4). These mixtures were incubated at 37°Cfor the appropriate time and the residual glutathioneconcentration determined as described by Woodward andFry"29 Mei 1971 S.-A. MEDIESE TYDSKRIF(Byvoegsel-Suid-Ajrikaanse Tydskrij vir Laboratorium- en Kliniekwerk)571LKW 515'5± 3·0 (4)is a racemic mixture of optically active isomers and ishydrolysed to near-completion under the above experi-mental conditions, it further appears that this reaction isnot stereo-specific for one optical isomer in so far assubstituents at they-position of the lactone ring are con-cerned. It is therefore more likely that enzymatic hydro-lysis of the lactones is controlled by nucleophilic attackby the enzyme on the carbonyl carbon atom. Due to theconjugation effect of the a/3 double bond the carcinogeniclactones would have a relative higher electron densityat the carbonyl carbon atom than the saturated lactonesand would therefore be relatively resistant towards nucleo-philic attack by this enzyme.As far as the further metabolism of the resulting 4-hydroxy fatty acids are concerned, it was previouslyshown that the saturated 4-hydroxy fatty acids are con-verted to their corresponding saturated fatty acids by ratliver slices but that this does not occur in the case oftheir a/3-unsaturated derivatives.llOn the other hand conjugation with glutathione occursonly with the unsaturated lac tones. From the resultsgiven in Table I it is clear that substituents at the y-position have a considerable effect on the extent of thereaction. Since the ratio of the lactone concentration tothe amount of liver from which the liver fractions wereprepared was the same in the glutathione conjugationexperiments as in the experiments on hydrolysis, it fur-thermore appears that the saturated lactones are moreeasily and rapidly metabolized by hydrolysis than theira,B-unsaturated analogs are by hydrolysis and conjugationwith glutathione.No metabolites were detected by thin-layer chromato-graphy employing cyclohexane-ethyl acetate (2: 8 v Iv) assolvent system of ethyl acetate extracts obtained afterincubation of the unsaturated lactones with the micro-somal hydroxylating system described by Boyland et al.12We wish to thank the South African Council for Scientificand Industrial Research for financial support for this study.REFERENCES1. Haynes, L. J. (1948): Quart. Med. Rev., 46, 46.2. Dickens, F. and Jones, H. E. H. (1961): Brit. J. Cancer, IS, 85.3. Idem (1963): Ibid., 17, lOO.4. Idem (1963): Ibid., 17, 691.5. Idem (1965): Ibid., 19, 392.6. Boyland, E. and Chasseaud, l. (1967): Biochem. J., 104, 95.7. Haynes, L. J. and Jones, E. R. H. (1946): J. Chem. Soc., Parr 2, 954.8. Goddu, R. F., Le Blanc, N. F. and Wright, C. M. (1955): Anal.Chem., 27, 1251.9. Woodward, G. E. and Fry, E. G. (1932): J. BioI. Chem., 97, 465.10. Fishbein, W. N. and Bessman, S. P. (1966): Ibid., 241, 4835.11. De Kock, D. H. and Raubenheimer, H. G. (1969): S. Afr. Med. J .•43, 959.12. Boyland, E., Kimura, M. and Sims, P. (1964): Biochem. J., 92, 631.TABLE 1. PERCENTAGE y-LACTONE METABOLIZED BY LIVERPREPARATION in vitro (INCUBATION TIME: 20 MINUTES)%Conjugationwith glutathioneooo7'2± 1-3 (4)y-lactone % Hydrolysisy-butyrolactone 73 ± 6·5 (3)y-valerolactone 83 ± 6·2 (3)y-isocaprolactone 64 ± 2·4 (3)y-crotonolactone 24± 9·3 (4)4-hydroxypent-2-enoic acidy-Iactone 14 ± 1·6 (5)4-hydoxy-isohex-2-enoicacid y-Iactone ·22±12 (5) 1·6±0·3 (5)Figures in parent~leses indicate the number of rats utilizedIn Table I the amounts of lactone metabolized by hy-drolysis and conjugation with glutathione are given. Fromthese results it is clear that the a/3-unsaturated y-lactonesare much more slowly enzymatically hydrolysed than thesaturated lactones. Since enzymatic hydrolysis is inhibitedby the sulphydryl reagent, p-cWoromercuribenzoate,'O anda, the sulphydryl group reacts with unsaturated lactones,the possibility that the slower hydrolysis of the latter maybe attributed to substrate inhibition was studied. By em-ploying lactone V as substrate and setting up a Line-weaver-Burke plot, a straight line was obtained, indicatingthat no substrate inhibition occurs. In this experimentthe rates of enzymatic hydrolysis at different substrateconcentrations were determined after 5, 10 and 15 minutesof incubation. The rates were linear for at least 10minutes.The relative small differences between the amounts oflactone hydrolysed in the saturated and unsaturatedgroups of lactones respectively are evidence that stericfactors due to the y-substituents do not have a significantinfluence on enzymatic hydrolysis. Since y-valerolactoneRESULTS AND DISCUSSIONIt was confirmed by inhibition studies with EDTA andp-chloromercuribenzoate as well as subcellular localiza-tion experiments that the decrease in lactone concentra-tion by incubation with the 10 000 G supernatant of arat liver homogenate is due to hydrolysis by the y-lacto-nase described by Fishbein and Bessman.'o The decreasein glutathione concentration when incubated with the y-lactones in the presence of the 120000 G rat liver super-natant fraction is due to conjugation of glutathione withthe latter compounds, catalysed by the enzyme describedby Boyland and Chasseaud." The latter reaction is pro-bably addition of the sulphydryl group of glutathione tothe a/3 double bond of the lactones.SOCIETY FOR ENDOCRINOLOGY, METABOLISM AND DIABETES OF SOUTHERN AFRICA:ABSTRACTS OF PAPERSThe following are abstracts of papers presented at the 10th Meeting of the Society for Endocrinology, Metabolism and Diabetesof Southern Africa held at the Athenaeum, Newlands, Cape, on 23 and 24 September 1970, during the 7th Biennial ScientificCongress of the Association of Physicians of South Africa:EFFECT OF INSULIN ON SODIUM TRANSPORT ANDWATER MOVEMENT ACROSS ISOLATED TOAD SKINR. J. DOUGLAS, L. C. ISAACSON AND R. RABKIN, Departments ofPhysiology and Medicine, University Dj Cape TownThe effects of ox insulin (Wellcome) and glucagon-free insulin(Liily Research Laboratories) on the transport of Na and wateracross toad skin (Xenopus laevis) were investigated. Na trans-port was quantitated by measurement of the short-circuitcurrent (SCC); water movement in response to an osmoticgradient was observed directly. All toads were pretreated withdeoxycorticosterone acetate.Of 18 skins challenged with crystalline insulin (25 - 200 mU Im}), 12 responded after a latent period of up to 30 minutes,with a prolonged rise in SCC. In 9 instances, this was ac-7

Metabolism of Gamma-Lactones by rat liver preparations in vitro

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Metabolism of Gamma-Lactones by rat liver preparations in vitro

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