EFFECT OF VITAMIN E ON MEMBRANES OF THE INTESTINAL CELL BY I. MOLENAAR, F. A. HOMMES, W. G. BRAAMS, AND H. A. POLMAN CENTER FOR MEDICAL ELECTRON MICROSCOPY AND DEPARTMENT OF PEDIATRICS, UNIVERSITY OF GRONINGEN, SCHOOL OF MEDICINE, GRONINGEN, THE NETHERLANDS Communicated by Emil L. Smith, September 12, 1968 Despite extensive research, many aspects of the role of vitamin E in metabolism are still uncertain. The studies by Martius' and Schwarz2 have yielded evidence for a specific role of vitamin E in redox reactions, while Tappell and Horwitt4 have given strong support for the antioxidant action of vitamin E as discovered by Olcott and Matill.' Vitamin E deficiency in man is rare, and its clinical picture is not well delineated. In patients suffering from prolonged steatorrhea with its associated impaired digestion or absorption of fats and fat-soluble vitamins, symptoms of vitamin E deficiency are sometimes encountered. Then a decreased erythrocyte-survival time6 and an increased hemolysis of erythrocytes in the hydrogenperoxide test' is found. Among the conditions in which the resorption of fats is disturbed, abetalipoproteinemia, an inborn error of metabolism, is a severe but an infrequent disorder.8 In two patients suffering from this disease and initially having low vitamin E levels of the blood normalizing upon vitamin E treatment, abnormal cellular ultrastructure was found, the description of which is the subject of this communication. Materials and Methods.-Two patients with abetalipoproteinemia and aged 4 years and 3 months, respectively, to be described in detail elsewhere, were studied. During these studies the patients received a constant diet free of natural fats but containing triglycerides of medium-chain fatty acids. These triglycerides, supplemented with corn oil as a source of the essential fatty acid linoleic acid (in an amount of 1 gm of corn oil per kilogram of bodyweight), were given as an emulsion in fat-free milk. This was the only source of fats available to the patients. Plasma vitamin E levels were determined according to the method of Lehman.9 Peroxide erythrocyte hemolysis tests were performed as described by Horwitt.6 Erythrocyte lifespan was determined with the "Cr method.'0 Jejunum biopsies were taken with the Crosby capsule," fixed immediately in 2% glutaraldehyde, buffered at ph 7.4 with phosphate buffer, and divided into small fragments. These were postfixed in 1% osmium tetroxide in Veronal acetate buffer, dehydrated in alcohol and via propylene oxide embedded in epon. The area of interest for electron microscopy was selected in sections 0.5 M thick and stained with crystal violetbasic fuchsin. Ultrathin sections were stained with lead citrate and uranylacetate, and photographed with a Philips EM 200 electron microscope. Results.-Parallel studies were made of erythrocyte membrane behavior and jejunal cellular ultrastructure. In Table 1 the pertinent data on plasma vitamin E levels, peroxide hemolysis tests, and half-life times of erythrocytes are summarized. Initially, the patients did not receive extra vitamin E except for a very small amount in the corn oil. In both patients, the increased hemolysis in the peroxide hemolysis test and the decreased half-life time of the erythrocytes normalized upon vitamin E treatment (cf ref. 6), whereas the plasma vitamin E level rose from 0.30 to 1.35 mg per 100 ml (Table 1). 982
VgOL. 6 1, 1968 BIOCHEMISTRY: MOLENAAR ET AL. 983 TABLE 1. Data from peroxide hemolysis test, vitamin E in plasma, and half life of erythrocytes. Hemolysis (%) Vitamin E in peroxide in plasma hemolysis Half life of erythrocytes (mg/100 ml) test* (days) Patient I 0.30 10.6 12.5 1st jejunum biopsy (age 4 years) 0.60-2nd " " 1.35 1.0 19 3rd " " Patient II 0.50-1st " " (age 3 months) 0.93 - - 2nd " 0.85 1.4-3rd " * Normal controls yielded an average of 0.7% hemolysis. Jejunal tissue was studied at three stages of vitamin E treatment: (a) before vitamin E treatment started-the patients had already received the constant diet for five months (first biopsy); (b) after the plasma vitamin E level had reached half the normal value, which required ten weeks of treatment (second biopsy); and (c) after the plasma vitamin E level had normalized, which took four months (third biopsy). Similar results were obtained with both patients. For reasons of space, however, only the micrographs of patient I are shown here. The macroscopical aspect of the villi was normal. Applying light microscopy, the integrity and morphology of the epithelial cells suggested the existence of a healthy mucosa. The electron microscope, however, disclosed in the first biopsy the absence of a basal feature of cellular ultrastructure: no membranes were visualized, at least not in positive contrast (Fig. 1). This was very striking in the mitochondria of the epithelial cells, showing "negatively stained" membranes surrounded by an electron-dense matrix (Fig. 2). The area of the cell where normally the rough endoplasmic reticulum can be found contained ribosomes arranged as expected in normal cells (e.g., in linear array) but without visible membranes. The perinuclear cisterna was present but without delimiting membranes. As the only exception, the plasma membrane was rather clearly visible, especially on the microvilli. Occasionally, very weakly stained Golgi membranes were also observed (Figs. 3 and 4). Vitamin E treatment, causing a rise of the vitamin E level of the blood, gave a dramatic change in the electron microscopic image of the second and third biopsies. After ten weeks, but even more clearly after four months, the epithelial cells showed their membranes, which stood out in normal contrast relations. The mitochondrial membranes seemed to respond earlier than the membranes of the endoplasmic reticulum. Finally, a completely normal cellular ultrastructure was observed; only the number of intracellular vacuoles seemed to be higher than normal (Fig. 5). In the underlying connective tissue, the cells appeared the same but in less spectacular fashion. Discussion.-The first finding to be discussed is the peculiar aspect of the membranes. Although no membranes are apparent in the electron micrographs taken before vitamin E treatment was started, this certainly is no conclusive evidence of their absence. It is more appropriate to think in terms of existing but nonosmiophilic membranes; in this respect it is to be noted that the linear
984 BIOCHEMISTRY: MOLENAAR ET AL. PROC. N. A. S. FIG. 1.-Jejunal epithelial cell with parts of microvilli at the top from a patient with abetalipoproteinemia, resulting in hypovitaminosis E. Note the lack of membranes in general (25,000X ). FIG. 2.-Two jejunal epithelial cells of the same patient, connected by desmosomes (d). Mitochondria (m) show their matrix, but lack positively contrasted membranes (30,000X).
VOL. 61, 1968 BIOCHEMISTRY: MOLENAAR ET AL. 985 FIG. 3.-Jejunal epithelial cell of the same patient. Next to "membraneless" ribosomes, a perinuclear cisterna (pc) without delimiting membranes is depicted (30,OOOX). FIG. 4.-Jejunal epithelial cell of the same patient. Ribosomes are present in linear array, but without membranes in positive contrast. Only on the microvilli a weakly stained plasma membrane (pm) is visible (40,000 X ).
986 BIOCHEMISTRY: MOLENAAR ET AL. PROC. N. A. S. t :. : -4F -i.'" "'i,4. :1. A 'Itz4o '. Al FIG. 5.-Jejunal epithelial cell of the same patient after 4 months of vitamin E medication. The cells have a normal ultrastructural aspect; apart from a rather large number of vacuoles, membranes are visible in normal contrast (20,000X).
VOL. 61, 1968 BIOCHEMISTRY. MOLENAAR ET AL. 987 array of ribosomes as well as the morphology of the mitochondrial matrix strongly suggests the presence of such membranes. It is known that membranes, notably mitochondrial membranes, contain a high amount of unsaturated fatty acids,"2 from which compounds membranes in ultrathin sections derive their contrast to a major extent.'3 Consequently, low contrast in membranes could be caused by a relatively low content of these fatty acids in the membranes. In this connection it is of interest to draw attention to the work of Morgan and Huber,'4 who studied loss of lipid during different fixation procedures. They found by chemical analysis for lung tissue, which is known to contain a relatively high content of saturated fatty acids, a considerable loss of lipid during osmification. A second finding is that all membranes are not affected in the same way. Relatively, the plasma membrane is the least vulnerable; to a lesser extent, this is true for the Golgi membranes. Much work has been done on the influence of fatty acid composition on the physiology of membranes. Much less is known about the factors determining the composition of the membrane in terms of the relative content of saturated versus unsaturated fatty acids. In bacterial membrane-lipids the kind of fatty acid depends upon the culture medium and growth phase. In late logarithmic phase the proportion of cyclopropane acids was greatly increased at the expense of the unsaturated acids.'5 In our material it has been shown that administration of unsaturated fatty acids did not enhance the contrast in cellular ultrastructure; only after vitamin E had been added to the diet did the membranes become apparent. This leads to the conclusion that vitamin E plays a decisive role in membrane metabolism. In this connection it should be noted that only membrane metabolism is at stake in the gut epithelium; its cells are renewed every four days! This can form a locus minoris resistentiae, where shortages in this respect will appear readily. There are two possibilities: (1) Vitamin E is a factor in the biosynthesis of membranes, particularly in the incorporation of unsaturated fatty acids; (2) vitamin E inhibits the breakdown of fatty acids of the membrane. This would be consistent with the previously mentioned concept of the antioxidant action of vitamin E. As to the anabolic or "catabolism inhibiting" role of vitamin E in membrane physiology, we have designed animal experiments on which we hope to report later. Summary.-In jejunal epithelial cells of two patients who initially had low vitamin E levels of the blood, intracellular membranes were not depicted in positive contrast with the electron microscope. Vitamin E treatment resulted in normal cellular ultrastructure. This leads to the conclusion that vitamin E plays a decisive role in membrane metabolism. The authors thank Prof. J. H. P. Jonxis and Dr. P. F. Ebels for critical and helpful discussion. 'Martius, C., Vitamins Hornwnes, 20, 457 (1962). 2 Schwarz, K., Vitamins Hormones, 20, 463 (1962). 3 Tappel, A. L., Vitamins Hormones, 20, 493 (1962).
988 BIOCHEMISTRY: MOLENAAR ET AL. PRoc. N. A. S. Horwitt, M. K., Borden's Rev. Nutr. Re8., 22, 1 (1961). Olcott, H. S., and H. A. Matill, Chem. Rev., 29, 257 (1941). 6 Horwitt, M. K., Vitamins Hormones, 20, 541 (1962). 7Horwitt, M. K., Am. J. Clin. Nutr., 4, 408 (1956). 8 Bassen, F. A., and A. L. Kornzweig, Blood, 5, 381 (1950). 9 Lehman, R. W., Methods Biochem. Anal., 2, 153 (1955). 10Mallison, P. L., and P. N. Veal, Brit. J. Haematol., 1, 62 (1955). 11 Crosby, W. H., and H. W. Kugler, Am. J. Digest. Diseases, 2, 236 (1957). 12 Kavanau, J. L., Structure and Function in Biological Membranes (San Francisco: Holden- Day, Inc., 1965), p. 97. 13 Millonig, G., and V. Marinozzi, in Advances in Optical and Electron Microscopy, ed. R. Barer and V. E. Cosslett (New York: Academic Press, 1968), vol. 2. 14 Morgan, T. E., and G. L. Huber, J. Cell Biol., 32, 757 (1967). 15 Rogers, H. J., and H. R. Perkins, Cell Walls and Membranes (London: Spon, Ltd., 1968), p. 361.