486 SHORT COMMUNICATION

13 E. B. FEICELSON, W. W. PFAFF, A. KARMEN ANU D. STEINBERG, f. C&n. Invest,, 40 (1961) 2x71.

14 H.G. WINDMUELLER AND A. E. SPABTH,.~YC!Z. Bicchim.Bio@ys., 122 (x967) 362. 15 J. T. BUCKLEY, T. J. DELAHUNTY AND D. RUBINSTEIN,&~. J.Biocksm., 46 f1968) 341. 16 D. S.ROBINSON AND A. SEAKINS, Biockana.Biophys. Acta, 62(1962) 163. 17 P. S. ROHEIM, L. MILLER AND W. A. EJXCR, J. Riot. Chews., 240(x965) 2994. 18 J. KRUH AND H. BORSOOK, J. Biol. Chew., zzo(rg56) 905. rg T. J. GRIBBLE AND H. C. SCHWARTZ, Biochim.Bio$hys. Acta, 103 1x965) 333" 20 A.I,GRAYZEL,P.HGRCHNERAND J.M.LoNDoN, Proc.NatE.Acad.Sci. U.S., 55 (1966) 650.

Received March z3rd, 1970

Biochim. Biapkys. A&, 210 (1970) 483-486

BBA 53278

The detection of oxidation in fi~~s~~~ preparations

If air is present during the preparation of liposomes from phospholipids of biological origin by sonication in an aqueous systeml, the K+ permeabilities measured for different preparations are not reproducible : the reproducibility is much improved by sonication under nitrogen. Phospholipids, and lecithin in particular, may undergo two forms of degradation in such an aqueous system. Hydrolysis may occur at the & ester linkage with the production of lysophosphatide and free fatty acids, or the unsaturated hydrocarbon chains may become oxidised. The hydrolysis to lysophos- phatide and fatty acid may be conveniently detected using thin-layer ~~lrornato~aphy on silica gel H plates with chloroform-methanol-7 M ammonia (230: go: 15, by ~01.)~ as solvent. A convenient and sensitive method for the detection of oxidation is described below.

It has been known for some time that polyunsaturated lipids may undergo free radical autoxidation in the presence of oxygen, and reaction mechanisms have been postulated for this reaction by FARMER AND SUTTON~, and discussed BY HOLMAN (ref. 4). The essential stages in the oxidation include bond mi~ation to give a con- jugated diene, followed by hydroperoxide formation. Ring formation and subsequent cleavage of the chain to give malonaldehyde and short-chain aldehydes and acids, complete the process. The initial step in the reaction is supposed to involve the loss of an rr: methylene hydrogen atom to give a free radical,

The methods available for determining the extent to which the sample has become oxidised could include the measurement of the peroxide value, the increase in absorption at 233 nm due to increasing diene conjugation, and the thiobarbiturate estimation of free and “bound’ ~lonaldehyde5. DAHLE et al. have discussed the measurement of these parameters for a range of polyunsaturated methyl esters?, and have shown that the diene conjugation and peroxide value provide a measure of the peroxidation of methyl linoleate (18:~) and higher polyunsaturated esters; whereas the thiobarbituric acid reaction yields no colour with linoleate, only doing so with the peroxidation products of linolenate (18 : 3) and higher polyunsaturated esters. The peroxidation of linoleate and higher polyunsaturated chains present in naturally occurring ~hospholipi~ may be detected2 therefore, by the measurement of either the diene conjugation or the peroxide value. LEAK has investigated the autoxidation

Biochina. Biofih~~. Acta, ZIO (1970) 486--&g

SHORT COMMUNICATIONS 487

Fig. I. The ultraviolet spectrum of liposomes prepared from purified egg yolk lecithin, at a concen- tration of 0.66 mM in abs. ethanol, recorded on a Pye Unicam SP 800 in r-cm quartz cuvettes.

of phosphatidyl ethanolamine and phosphatidyl choline, and has shown that changes occur in the ultraviolet spectrum at 235 nm and at 270-280 nm on exposure to oxygen, the change at 235 nm reflecting the increase in conjugated diene hydroperoxides. More recently HUANG~ has pointed out that the development of absorption maxima between 265 and 285 nm in the ultraviolet spectrum of phosphatidyl choline, in the form of sonicated liposomes, would appear to indicate the presence of conjugated triene chromophores produced by the oxidation of the fatty acid side chains. However, the appearance of such maxima indicate the peroxidation of fatty acids containing only three or more double bonds (i.e. linolenate and higher), whereas the increase in diene conjugation as judged by the absorbance at 233 nm, is applicable to linoleate and higher polyunsaturated chains. For the purpose of screeningliposome preparations for autoxidation during experimental procedures, the measurement of the change in absorbance at 233 nm is the most useful and convenient, as diene conjugation repre- sents a very early stage in the reaction, and the measurement may be achieved very rapidly using modem recording UV spectrophotometers.

Ultraviolet spectra were recorded for the liposome samples under the following conditions : about 2 ,umoles of lecithin, or other phospholipid, as a liposome prepara-

Fig. 2. Lecithin concentration against absorbance at various wavelengths, with abs. ethanol as solvent.

Fig. 3. Oxidation index against time for a liposome preparation sonicated in the presence of either air or nitrogen.

Biochim. Biofihys. Acta, 210 (1970) 486-480

488 SHORT COMMUNICATIONS

tion were added to 3 ml of absolute ethanol. This amount of ethanol will dissolve 0.1 ml of aq. IOO mM KC1 solution containing the dispersed liposomes. Typical spectra (recorded on a Fye Unicam SP 800) are shown below for a fresh and an oxidised sample.

For convenience an “oxidation index” is required. However, because of instru- mental limitations in the region of 200-210 nm, the peak observed at 205-210 nm is not a reliable datum for comparison with the absorption at 233 nm. This peak at 205-210 nm is almost certainly a shifted and distorted carbonyl group absorption resulting from instrumental limitations at this end of the spectrums+*.

The peak height, although concentration dependent, does not obey the Beer- Lambert law, and because of this a family of curves was plotted of absorbance at various wavelengths against concentration of lecithin. The requirements for a reliable datum wavelength are that the absorption at this wavelength should depend linearly on the amount of lecithin used, and should be far enough away from 233 nm so that oxidation does not cause interference. In the case of the SP 800 used, the lowest wavelength that fulfilled these criteria was found to be 215 nm. It is worth empha- sizing that the exact value for this wavelength will vary from instrument to instru- ment, depending on the extent to which stray light is a problem. An “oxidation index” was defined, therefore, as the ratio of the absorption at 233 nm to the absorption at 215 nm (oxidation index = A,,, n,/A,,, nm).

When a liposome preparation was sonicated (Kerry’s Ltd. KB 8011 Bath sonicator) in the presence of air the index increased with time, whereas in the presence of nitrogen it did not increase. Ultraviolet light, manganese, iron, and cobalt ions were found to increase this rate of change, and this is consistent with a free radical mechanism of oxidation. Sequential analysis of the fatty acid composition of the lecithin during sonication, showed that the polyunsaturated acids were destroyed preferentially, and that the effect was most marked when there were three or more double bonds present in the hydrocarbon chain. This finding is to be expected from the postulated reaction mechanism 81~. The results are summarized graphically below in Figs. 4 and 5.

Fig. 4. Percentage of the original amount of fatty acid esterified against time of sonication, in the presence of either air or nitrogen. Fatty acids determined as methyl esters by gas-liquid chromato- graphy on polyethylene glycol adipate at 197’. Palmitate as reference; palmitate to stearate ratio remainsconstant.o,18:o; x,18:1; +, x8:2; @,18:3;0, 20:4; l ,zz:5(a);C?,22:5(b):~,zz:6.

Fig. 5. Percentage of the original amount of fatty acid esterified, after 4.5 h sonication in the pres- ence of air, against the number of double bonds present in the acid.

Biachim. Biophys. Acta. 210 (1970) 486-489

SHORT COMMUNICATIONS 489

It has been established that an increase in the “oxidation index” is associated with increased permeability properties in a preparation of liposomes. However, this does not imply the converse that increased permeability properties are always asso- ciated with a raised “oxidation index”. Increased permeability may also be caused by other variables such as hydrolysis. Preliminary data suggest that there is a quan- titative correlation between a raised Kf permeability and an increased “oxidation index” (S. M. JOHNSON, personal communication).

In conclusion, therefore, the index A 233 ,lm/AZ1S ,,,,, provides a rapid and sensitive method for detecting oxidation in liposome preparations during their preparation or subsequent manipulation. The lower limit of detection is conservatively estimated as 0.02 absorbance unit, equivalent to approx. 0.1% oxidation, assuming reasonable values for the relevant extinction coefficients.

Particular thanks are due to Dr. A. D. Bangham, in whose laboratory this work was carried out, for many helpful and instructive discussions. Financial support from Unilever Limited is gratefully acknowledged.

Institute of Animal Physiology, Agricultural Research Council, Babraham, Cambridge (Great Britain)

R. A. KLEIN

I S. M. JOHNSON AND A. D. BANGHAM, Biochim. Biophys. Acta, 193 (1969) 82. 2 W. D. SKIDMORE AND C. ENTENMAN, J. Lipid Res., 3 (1962) 471. 3 E. H. FARMER AND D. A. SUTTON, J. Chem. Sot., (1943) rug. 4 R. T. HOLMAN, in R. T. HOLMAN, W. 0. LUNDBERG AND T. MALKIN, Progress in the Chemistry

of Fats and Other Lipids, Vol. 2, Pergamon Press, London, 1954. p. 51. 5 L. K. DAHLE, E. G. HILL AND R. T. HOLMAN, Arch. Biochem. Biophys., g8 (1962) 253. 6 C. H. LEA, in G. POPJAK AND E. LE BRETON, Biochemical Problems of Lipids, Butterworths,

London, 1956, p. 81. 7 C. HUANG, Biochemistry, 8 (1969) 344. 8 J. R. EDISBURY, Practical Hints on Absorption Spectrometry (Ultraviolet and Visible), Hilger

and Watts, London, 1966, Chapter 12. g W. SLAVIN, Anal. Chem., 35 (1963) 561.

Received March 23rd, rg7o

Biochim. Biophys. Acta, 210 (1970) 486-489

BBA 53280

The subcellular distribution of triglyceride synthetase in the intestinal mucosa

Recently several reports have appeared suggesting that the brush border fraction may be a major site for the synthesis of triglycerides in the intestinal mucosa (refs. 1-4). In contrast to the above observations were the previous reports of BRIND- LEY AND HOBSCHER~ in which the various subcellular fractions were carefully moni- tored by enzyme markers and it was concluded that the enzymes responsible for triglyceride biosynthesis via the monoglyceride pathway were confined primarily to

Biochim. Biophys. Acta, 210 (1970) 489-492