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Fig. 1, Nuclear-pore-complex-rich fraction from a 0.25-1 .Owsucrose density gradient of ultrasonicated Triton X- 1 00-extracted rat liver nuclear envelope Negatively stained with 2 % ammonium molybdate (pH7.0). Magnification x 30000.

rich in nuclear-pore complexes. This suggests that a significant number of pore complexes may be disrupted by ultrasonication, the smaller breakdown products then being located in the less-dense gradient fractions. The nuclear-pore-complex-rich fraction nevertheless possesses a significantly different polypeptide composition from the initial Triton X-100-extracted nuclear envelope. Gall, J. G. (1 967) J . Cell B i d . 32, 391-400 Harris, J. R. (1974) Philos. Trans. R . Soc. London, Ser. B 268, 109-117 Harris, J. R. (1977) in Methodological Surveys, Vol. 6 1 Membranous Elements and Mocernent of Molecules: Techniques (Reid, E., ed.), pp. 245-250, Horwood, Chichester Harris, J. R. & Marshall, P. (1977) Micron 8, 217-219 Harris, J. R. & Milne, J. F. (1974) Biochent. Soc. Trans. 2, 1251-1253 Yoo, B. Y.& Bayley, S. T. (1967)J. Ultrastruct. Res. 18,651-660

The Fate of Liposomes in the Rat Small Intestine DAVID A. WHITMORE and KENNETH P. WHEELER Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNl 9QG, U.K. The oral administration of insulin entrapped in liposomes, or of insulin in the presence of ‘empty’ liposomes, produces a hypoglycaemic effect in rats (Patel & Ryman, 1976, 1977a). Entrapped insulin is protected from proteolytic digestion (Patel & Ryman, 1977b), but the reported absorption of intact liposomes from the gastrointestinal tract (Dapergolas & Gregoriadis, 1976) has not been confirmed (Patel & Ryman, 1977b). We have therefore studied the absorption of liposomes by the intestine in vitro, using a simple everted-sac preparation, and also examined the stability of liposomes in the presence of bile, protein and lipids.

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Table I . Loss of zzNafrom liposomes as a measure of liposome disruption in the presence of various compounds Liposomes containing trapped 22Na were prepared as described in the text. Samples (0.25ml; 6.25pg of lipid) were incubated in a final volume of 1 ml of buffer solution (pH6) containing various other compounds, as indicated, for 20min at 37°C. The proportion of liposomal ZZNawas then determined by fractionation through a column of Sephadex G-50. (In experiments with everted sacs the final mucosal volume was 10 ml and the liposomal lipid was 0.64pmol/ml.) Fraction of "Na in liposomes (%) Addition None Everted sac Bile (0.01 %, v/v) Bile (0.1 %, v/v) Bile (1 %, v/v) Bile ( 10%. v/v) Bovine serum albumin (50mg/ml) Phosphatidylcholine (50 mg/ml) Glycerol (10 %, v/v) Oleic acid (100mg/mI) ( d ) DEAE-Sephadex (30mg/ml)

Control 89 74 82 72 14 0 89 97 91 0 88

Plus 30 % bile 0 -

17 24 0 0 27

Everted sacs of jejunum were prepared and incubated as described by Whitmore et al. (1979). Liposomes were made from phosphatidylcholine, cholesterol and phosphatidic acid (molar proportions 10:2: 1) by sonication in either Krebs phosphate buffer, pH 6 , or 20m~-4-morpholine-ethanesulphonic acid/NaOH buffer, pH 6 at 20°C. The final concentration of lipid was 25pg/ml. ZZNaClwas added to the buffer solutions so that after separation through Sephadex G-50 the liposomes contained 3.6nCi of z2Na/pmol of lipid P. The everted sacs were incubated in 9ml of Krebs phosphate buffer plus 1ml of liposomes for 20min at 37°C. The final concentration of liposomal lipid was 0.64pmol/ml. The mucosal and serosal fluids were then fractionated on Sephadex G-50 and assayed for z2Na radioactivity. Liposomes (0.25 ml, containing 1.6pmol of lipid) were also incubated with 0.75 ml of buffer solution containing various compounds intended tn be characteristic of the digestive milieu (Table 1) or with rat bile that had been previously equilibrated with each compound. The distribution of 22Nabetween liposomes and incubation medium was determined as above. No significant amount of radioactivity was detected in the serosal fluid from the everted sacs; recovery from the mucosal solution was 99 f 1 % (s.E.M., n = 5). Thus neither intact liposomes nor free solute could have been absorbed from the mucosal solution except in quantities too small to be detected by our techniques. The presence of the sacs caused an increased loss of "Na from liposomes (Table la) that might have been the result of residual phospholipase activity. The presence of bile at a concentration greater than 1 % (v/v) produced complete loss of liposomal z2Na (Table lh); the other compounds tested had various effects (Table lc). The disruptive effect of bile was, however, modified by either protein or phospholipid, and sequestration of bile salts by DEAE-Sephadex similarly prevented complete breakdown of the liposomes (Table Id). Hence the advantage of liposomal entrapment of solutes for oral administration appears to be limited to a potential protective action, if the solute is sensitive to attack by digestive enzymes. Also, it seems to be feasible that such protection is a f u n d o n of 1979

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the added lipid rather than the actual entrapment of solute inside liposomes, since the action of phospholipases as well as of bile must be taken into account. We thank the Science Research Council for a C.A.S.E. award and Upjohn Ltd. for some financial support. Dapergolas. G. & Gregoriadis, G. (1976) Lancet ii, 824-827 Patel, H. M. & Ryman, B. E. (1976) FERS L e f f .62, 60-63 Patel, H. M. & Ryman, B. E. (19770) Biochem. Soc. Truns. 5,1054-1056 Patel, H. M. & Ryman, B. E. (19776) Biochem. SOC.Trans. 5,1739-1741 Whitmore, D. A , , Brookes, L. G. &Wheeler, K. P. (1979)J. Phurm. Pharmacol. 31,277-283

Microvesicles are Released from Human Erythrocytes Aging at 4°C in Heparin and Citrate/Phosphate/DextroseAnticoagulants MARK WHITE,* MARTIN G . RUMSBY* and L. A. DERRICK TOVEY? *Department of Biology, University of York, Heslington, York YO1 5 D D , U.K. and ?Regional Blood Transfusion Centre, Bridle Path, Leeds, LS15 7TW, U.K.

The discocyte-spherocyte shape change that occurs during the aging of human blood stored in uitro for transfusion at 4°C in acid citrate/dextrose (ACD) is now a well-defined process (Brecher & Bessis, 1972; Longster et al., 1972). Concurrent with the formation of spheroechinocytes 1 (Bessis, 1972) in the shape transformation is the release of membrane as microvesicles to the plasma (Rumsby et a[., 1977; Trotter, 1978). These microvesicles are filled with haemoglobin and contain many of the normal erythrocyte membrane proteins with the notable exception of spectrin. This present communication reports our observations on the aging of human blood in heparin and citrate/phosphate/ dextrose (CPD) anticoagulants in relation to microvesicle release. Individual blood donations were taken directly from donors into three pre-sterilized bottles, one each for heparin, acid citrate/dextrose and citrate/phosphate/dextrose. The blood samples were well mixed and portions were withdrawn under sterile conditions ; bottles were then stored at 4°C. At weekly intervals the bottles were mixed and portions of blood were taken for analysis. Cells were sedimented by centrifugation at lOOOg for 10min. A sample of the plasma was taken for haemoglobin assay. Remaining plasma was centrifuged first at 2500g for lOmin and then at lOOOOOg for 60min to recover microvesicles. Microvesicles in the l00OOOg pellet were measured quantitatively by assaying for haemoglobin. Erythrocyte shape was monitored by scanning electron microscopy with a Cambridge 600 instrument. Cells were fixed for microscopy by the procedure of Bessis & Weed (1972). Plasma glucose concentrations were measured with a Boehringer test kit [Boehringer Corp. (London) Ltd., kit no. 1391061. The results in Fig. 1 show that the release of total haemoglobin from cells into plasma is almost 1 0 0 times greater with heparin as anticoagulant compared with acid &rate/ dextrose or citrate/phosphate/dextrose. By week 3 the plasma haemoglobin concentration of blood stored in heparin was over 3000mg/100ml whereas for acid &rate/ dextrose and citrate/phosphate/dextroseit was only 3040mg/100ml. The proportion of spheroechinocytes 1 in aging blood samples was much larger in heparin-stored samples; 60% of the cells were this form for heparin compared with 20-30% for citrate anticoagulants. Heparin blood samples at week 4 also contained ‘ghosts’, which were not present in acid citrate/dextrose or citrate/phosphate/dextrosesamples. Microvesicles containing haemoglobin were detected in the plasma of blood aging in both heparin and citrate/phosphate/dextrose(Fig. 1). Though heparin-aged samples had a much higher proportion of cells as spheroechinocytes 1 by week 4, the rate of microvesicle release was not much higher than for acid citrate/dextrose or citratephosphate/dextrose samples (Fig. 1). Analysis of the microvesicle membrane by polyacrylamide-gel electrophoresis showed that heparin-aged vesicles lacked the major spectrin bands and that, essentially, the polypeptide pattern was similar to results

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