EXPE~MENTAL
Ultrastructural
AND
MOLECULAR
PATHOLOCY
24, 236-243
and Stereological Study on Liver Mitochondrial M.
Received
May
(1976)
on the Effect Membranes
of Vitamin
E
FRIGC 1 AND H. P. ROHR 2
13, 1975,
and in revised
form
October
1, 1975
The quantitative composition of hepatocytes in mice was analyzed stereologically in vitamin E deficiency, after vitamin E supplementation and feeding of a standard diet. No difference between the three groups,of five animalseach,could be observed descriptively. Stereologically, the vitamin E deficiency group showed a significant increase of the surface density of the inner nlitochondria~ membrane. A model-like calculation of the “center to center distance” of cytochrome molecules revealed mean distances of 180-230 d in the control group, whereas, in the vitamin E deficiency group, these distances were nearly doubled.
1. INTRODUCTION The function of vitamin E as a biological antioxidant has been discussed from various points of view (Tappel, 1962; Henn and Thompson, 1969; Lucy, 1972). The antioxidant hypothesis has been confirmed by the possibility of repIacing vitamin E for certain functions, in vitro and in Vito, by synthetic antioxidants. Other studies (Green et at. 1967; Diplock et al. 1968, 1971) showed that the prevention of autoxidation of lipids may not be a direct function of vitamin E. Caygill et aZ. (1971) suggested a protective action of vitamin E for reduced selenium, The concentration and the intracellular distribution of selenide, which like vitamin E (Gloor, 1970; Wiss et al. 1962) is mainly found in mitochondria (Caygill et al. 1971) are strongly influenced by vitamin E. Lucy (1972) suggestsstabilization of membranes by vitamin E as an explanation for the increased vitamin E requirement where there is a high content of unsaturated fatty acids in the food. This stabilization is apparently caused by a physicochemical interaction between the phytyl side chain of vitamin E with polyunsatmated acyl chains of fatty acids. The stabilizing effect of vitamin E on the erythrocyte membrane was proved by Friedman et al. (1958) and Weiser (1969) by means of the hemolysis test. Molenaar et al. (1970) and Vos et al. (1972) observed, in ducklings and human biopsies, alterations in the electron microscopical contrast of mitochondria1 membranes from the epithelial cells of the jejunum, which were dependent on vitamin E. Riede et al. (1972a, b) observed in the hepatocytes of rats after chronic vitamin E deficiency an increase of the surface of the inner mitochondrial membrane by using ultrast~c~ral stereology. The occurrence of secondary symptoms in advanced vitamin E deficiency cannot be excluded. In the
Copyright ~11 rights
Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
LIVER
MITOCHONDRIA
AND VITAMIN
E
237
present study, an attempt was therefore made to determine quantitatively primary ultrastructural alterations of the mouse liver in vitamin E deficiency by assessing the vitamin E deficiency state (hemolysis test). 2. MATERIALS Experimental
AND
METHODS
Design
Male hybrid mice C57BLJ//CBA/J with an initial weight of 20-24 gm were divided into three groups of ten animals each: group 1 (ED) was fed a vitamin E-deficient ration ( Schwieter et al. 1966), group 2 (ES) was fed the same ration supplemented with 100 IU dLa-tocopherol acetate and group 3 (CD) obtained a commercial standard diet (NAFAG 869). A modified hemolysis test (Friedman et al. 1958; Leonard and Losowsky, 1967; Weiser, 1969), with a dialuric acid concentration of 4 mg/liter, was used to characterize the vitamin E deficiency. After a trial period of 17 weeks, tissue samples from the left liver lobule were taken under anaesthesia for stereological examination. For electron microscopy and the stereological model we refer to Rohr et al. ( 1976) and Weibel ( 1969, 1973). 3. RESULTS Hemolysis Test and Deficiency Symptoms
Table I shows the values from the hemolysis test with two different dialuric acid concentrations. In all tests, the animals from the vitamin E-deficient group showed a high hemolysis rate ( >92% ), which was significantly different from that of groups 2 and 3 (P < 0.05). With 16 mg dialuric acid/liter, the hemolysis rate is high also for groups 2 and 3 (71.5%, 70.4%, group 1 97.4%), but there is, however, no difference between groups 2 and 3. The low dialuric acid concentration of 4 mg/l results in a significant difference between groups 2 and 3 (P < 0.05). TABLE Hemolysis
Test in Animals of Group Vitamin E), and Group
Dialuric concentration Hemolysis
Mean Standard
1 (Vitamin E Deficiency), Group 2 (Supplemented 3 (CD) a Week Prior to Conclusion of Trial
Vitamin E deficiency
Group acid mg/l rate
error
in
70
(SM)
I
Vitamin E supplementation
different different
from from
CD
4
16
4
16
4
16
94.6 92.6 96.9 98.6 98.6
98.5 98.6 92.3 98.7 98.8
31.8 40.2 38.5 34.9 41.4
64.6 61.3 81.5 81.0 68.9
26.5 20.0 34.7 22.9 29.2
74.6 59.7 81.5 74.6 61.5
96.3” 1.17
97.4” 1.27
37.4* 1.76
71.5 4.17
26.7 2.54
70.4 4.19 __-
0 Significantly b Significantly
with
groups group
2 and 3, P < 0.05. 3, P < 0.05.
number
volume surface volume surface
Mitochondria
RERb RERb SERc SERC
cma/cnlJ m2/cm3 em3/cm3 ma/cm3
cms3
cmS/cm3 om8/cmz ema/cm3 cm3/cm3 mz/cm3 m*/cm3 cm-a
Unit
0 = Shape factor. a Significance in the l-test: 1, comparison P 6 0.05 at 8 degrees of freedom. b RER = Rough endoplaamic retkulum. * SER = Smooth endoplasmic reticulum.
volume volume volume volume surface surface number
Paramet~er
Hepatocyte Cell nucleus Cytoplasma ~~itoehondria Xlitochondria Mitochondria Xitochondria
___-.
Compart,ment
vitamin
‘5. t I 0; 0; 2;3; 0;
VW vvx &MC NW
0;
VVSEX SWEi
E supplementation/CD;
1; 0;
&RER
0;
V VREK
(@ = 1.4.;) NVM (j3 = 2.25) 0;
0;
&MO
3;
VVNH
Significancea
Values
VVR
Symbol
Stereological
0.3284 1.4214 0.0136 0.1908
Volume
Liver
E supplementation/vitamin
0.0258 0.133 0.0030 0.0432
0.0062
0.0084 0.0250 0.0091 0.0073 0.0227 0.0701 0.0102
SE
E deficiency
* 1012
0.0774.10’2
0.9176 0.0840 0.8336 0.1646 0.4674 1.3184 0.1292
?U
Vitamin
to the Unit
II
2, vitamin
Related
TABLE
0.302S 1.7328 0.0140 0.1464
0.0794
0.0543 0.1945 0.0033 0.0334
0.0064
0.0122 0.0139 0.0131 0.006 0.0667 0.1063 0.0105
SE
IlZ deficiency;
* 10’2
0.9010 o.osso 0.8130 0.1662 0.4426 0.9894 0.1320.10’2
m
Vitamin E supplementation
3, CD/vitamin
0.2834 1.1764 0.0174 0.3316
0.0894
0.0248 0.0740 0.0035 0.0887
0.0077
0.0108 0.0139 0.0092 0.0088 0.0256 0.0832 0.0128
SE
E deficiency;
* 10’2
0.8812 0.0620 0.8192 O.i666 0.3366 0.7726 0.1488~10’~
‘VL
CD
il
z:
G u
3 $j
LIVER
M~OCHONDRIA
AND ~TAMIN
TABLE Stereological Compartment
Parameter
E
239
III
Values Related to Unit Volume Cytoplasma of Hepatocytes Unit
Symbol
SiinifiC*nCe~
Vitamin defi&noy
E
Vitamin E supplementation m
CD m
SE
m
SE
SE
0.1906 0.5412
0.0095 0.0306
0.1940 0.5120
0.0079 0.0724
0.1992 0.4034
0.0088 0.0305
Mitochondrium
volume surface
Crn~/Ornf rn~/Crn~
Z$20
0:
gi&hondnum Mitochondr+n
surface vollmle
cm*/cm* m~/cm*
VVRER/VVC SYMC/ VW
z i3 ;
1.5260 0.3790
0.0290 0.0862
0.3532 1.1478
0.0615 0.109%
0.3390 0.9264
0.0287 0.1ooe
EiY SER:
surface volume surface
m~/mn* cm*/cm* m*/cmE
SVR&R/VVO vv8ER/vvo SVE~/V~O
of yj 0;
0.0160 1.6494 0.2214
0.1676 0.0035 0.0512
0.0166 2.0110 0.1698
0.2033 0.0037 0.037
0.02m 1.4092 0.3992
0.0873 0.0043 0.1096
5 Siiificanca in vitmnin E defkienoy, b RER = Rough c SER = Smooth
Student’s test: 1. comparison 3. CD/vitamin E deficiency, endoplasmio reticulum. endoplaamio reticulum.
vitamin
No difference in weight development served between the groups. Ultrastructural
E suppleme~~tion/CD, at 8 degrees of freedom.
P < 0.05
or behavior
2. vitamin
E s~ppiementation/
of the animals could be ob-
Aesdts
No differences between the three trial groups can be evaluated descriptively. The mitochondria are built up normally and the efectron density of the matrix is unchanged. Stereological
Results
The volume density of the mitochondria in the reference systems liver, hepatocyte and cytoplasm, does not show any significant difference between the three trial groups, control group (CD), vitamin E-supplemented group (ES), and vitamin E-deficient group ( ED). The main finding consists of ‘an increase of the surface density of the inner membrane of the mitochondria in the vitamin E-deficient group (Tables II and III). The surface density of the outer membrane of the mitochondria is only increased signi~cantly when compared with the CD group (Tables III and IV). Also the group supplemented with vitamin E showed an increased surface density of the inner mitochondrial membrane in comparison to the CD controls. When related to the unit volume mitochondria, these values are also greater in the deficient and supplemented groups compared to the CD group (Table IV). The volume density of the mitochondria (Tables II and III) as well as the mean single volume of mitochondria (Table IV), however, remain unchanged.
Stereological ParWleter
Mitoohondria: Outer membrane Outer membrane Inner membrane Inner membrane Mean volume (@ = 1.45)
:;;z~ surface surface volume
Tinit
m~/cm~ WQ m~/om: em’ tm0
n Significance in Student’s &teat: vibmin E deficiency; 3. CD/vitamin
Symbol
SVMO/VVM pMM/;~ v f$y/gNvy
1. clomp&son E deficiency;
TABLE IV Values of Mitwhondria Significnnce’
3; 3; 2;3; 1;2;3; 0;
vitamin
P 6 0.05
Vitamin deficiency
E
m
SE
2.6638 3.7544 8.0118 10.3816 1.3016
0.2086 0.4768 0.3419 0.3508 0.1128
E suppbmentation/C.D. at 8 degrees Of freedom.
Vitamin E supplementation m
2.6654 4.1022 6.9664 7.4934 1.2662
SE
0.3897 1.0630 0.6511 0.6221 0.1066
2. vitamin
CD
~ pn
2.0360 2.3404 4.6714 5.2394 1.1718
E supplementation/
SE
0.1911 0.2833 0.6097 0.4574 0.1639
FRIGG AND ROHR
240
TABLE II~~ueIl~
of a-Tocopherol on ~fitoehondrial Cytochromes (according
&011p
mg Mitochondrial protein/gm liver .-_-.--___-
__..-Standard Vitamin Vitamin a Data
diet (CD.) E supplementation E deficiency from
Table
V Proteins to Schwarq nMol
14.99 9.06 8.31
and on Various 1972)
Cytoohrome/mg chondrial protein
of Their
mito-
a
b
Cl
c
0.26 0.31 0.2.i
0.35 . 0.37 0.31
0.22 0.27 0.22
0.2.3 0.2.: 0.27
SYd~VC” (m”/cm3)
0.9264 1.1478 I.5260
III.
The volume and surface values of the rough and smooth elldopIasnlic reticulum show no changes in all three trial groups. 4. DISCUSSION The surface density of the mitochondrial inner membrane of the vitamin Edeficient group is increased significantly (Fig. 1) compared to the two control groups. The surface to volume ratio of the inner membrane of the mitochondria (Sv&Vvn) is higher in the vitamin E-deficient group than in the control group (Fig. 1). A swelling of the mitochondria with chronic vitamin E deficiency, which should result stereologically in a reduction of this ratio can, therefore, be excluded. Rather can an augme~~tation of the inner nlitochondrial membrane be assumed, since the volume density of the mito~hondria remains constant in all reference systems. A genuine enlargement of the surface of the mitochondrial membranes requires, on the one hand, the formation of membrane elements, as well as, on the other hand, the synthesis of membrane-bound enzymes. Membrane elements and %MC1UUM m2h3
T
ED
ES
1. Surface density of mitochondrial outer to the volume density of mitochondria (VW). FIG.
CD
ED
( SvMo)
ES CD
and
inner
( SvMa)
membranes
related
LIVER
MITOCHONDRIA
AND VITAMIN
TABLE
E
241
VI
Mean Distances (A) of Cytochrome Molecules in Animals with a Normal Diet (CD), in Animals Fed a Vitamin E-Deficient Diet with and without Vitamin E Supplementationa Group
Normal diet (C.D.) Vitamin
E supplementation
Vitamin
E deficiency
a The cytochrome concentrations
Cytochrome
A % A % A %
a
b
Cl
c
210.0 100.0 280.0 130.0 380.0 180.0
180.0 100.0 260.0 140.0 340.0 180.0
230.0 100.0 300.0 130.0 400.0 170.0
220.0 100.0 310.0 140.0 360.0 170.0
according to Schwarz (1972) were used for the calculations.
mitochondrial enzymes are mainly synthesized in the rough endoplasmic reticulum and, to a smaller extent (approximately 10% ), intramitochondrially ( Beattie, 1971). Under pathological conditions, however, it is conceivable that a distinction must be made between various proliferation types of mitochondrial membranes: 1. Membrane-bound enzymes and membrane elements are both formed proportionally to an increased extent. 2. Only membranes are synthesized. The formation of enzymes remains constant or is reduced. In such a case, the occurrence of membranes with a “diluted” enzyme content must be assumed. 3. The membrane formation is enhanced, the enzyme formation, however, more intensively in comparison to the membrane formation. There result membranes with a compacter enzyme content. Neither descriptively, stereologically, nor biochemically can a distinction be made between these three hypothetical types of membrane proliferation. Only a suitable correlation with stereological and biochemical data can furnish further elucidation (cf. Reith et al. 1973). Therefore, it was tried to confirm this evidence of a disturbed membrane integrity by means of a model-like calculation of the mean enzyme distances (center to center distance) on the mitochondrial inner membrane. According to Schwarz (1972), in vitamin E deficiency, the content of mitochondrial protein per gm liver tissue and cytochrome concentrations per mg mitochondrial protein are reduced (Table V). Since the arrangement of the groups in the studies of Schwarz (1972) corresponds to a great extent with the trial plan described here, the results of Schwarz (1972) were used to calculate the mean enzyme distances. For the calculation of the enzyme distances, we made the following assumptions (cf. Reith et al. 1973, and Rohr et al. 1976) : 1. The enzymes are distributed homogeneously in the inner membrane. 2. The density of the enzyme content should be expressed as mean area which may be assigned to one enzyme, respectively, as “center to center distance.”
242
FRIGG
AND
ROHR
The calculation of this mean “center to center distance” is explained by the following example (CD control group) : Cytochrome a : 0.26 n&fof/mg mitochondrial protein Mitochondrial protein: 14.99 mg/gm liver tissue Surface of the inner membrane 0.926 m2/cm3 liver tissue ( &MC/VVC ) : Cytochrome share in Iiver tissue: 3.898 nA4oI,/gm liver, corresponding to 23.478- 1Ol4 molecules Mean area (F) per molecule: 39.441 A Mean distance (d) of cytochrome a molecule assuming a hexagonal arrangement: d = 213 A It appeared that, in the CD control group, the mean distances of the cytochrome molecules lie in the range of 180-230 A. Reith et aE. (1973) calculate values of 260 A, whereas, according to Lehninger ( 1970)) the “center to center distances” of the respiratory units should amount to 230 A. In the heart muscle, the mean statistical distance of the cytochrome a molecule is 220 A (Klingenberg,
1970). Chronic vitamin E deficiency Ieads to nearly double the mean distances of the four cytochromes (Table VI). The vitamin E-supplemented diet, too, leads to an increase of 30-40s of these enzyme distances. These correlated biochemicostereological rest&s show that the formation of mitochondrial inner membrane is enhanced in vitamin E deficiency, whose cytochrome density, however, is reduced. These findings indicate that the formation of mitochondrial inner membrane increases in vitamin E deficiency states, the synthesis of the membranebound cytochrome, however, does not seem to be enhanced proportionaIIy. The stereologica1 findings as well as the model-like calculations of the mean “center to center distances” of the cytochrome molecules show that, in a chronic vitamin E deficiency state, structurally and thus functionally modified (defective) membranes are formed to a greater extent. Thus, the investigations of Schwarz (1972), Yeh and Johnson ( 1973) as well as Carabello (1974), which point to a disturbed membrane integrity with vitamin E deficiency, are further supported. REFERENCES BEATTIE, D. (kiABELL0, Biochem. CAYGILL, C. intracellular
S. (1971). The synthesis of mitochondrial proteins. Subcell. F. B. (1974). Role of tocopherol in the reduction of mito~hondrial
52, 679-688. P. J,, LUCY, J. A., and DIPLOCK, A. T. ( 1971). distribution
of the
different
oxidation
states
Biochem. NAD.
The effect of vitamin of selenium in rat liver.
1, l-23. Can. J, E on the Biochem.
3. 125,407-416. DIPLOCK, A. ‘I’., CAWTHORNE, M.
A., MAXWELL, E. A., GREEN, J., and BUNSAN, J. (I968). Measurement of Iipid peroxidation and ol-tocopherol destruction in vitro and in vioo and their significance in connection with the biological function of vitamin E. &it. J, N&r. 22,
405-472. DIPLOCK, A. T., BAUM,
H., and LUCY, J. A. ( 1971). The effect of vitamin E on the oxidation of selenium in rat liver. 3iochem. J. 123, 721-729. FRIEDMAN, L., WEISS, W., WHERRY, F,, and KLINE, 0. L. ( 1958). Bioassay of vitamin E by the dialuric acid hemolysis method. J. Nutr. 65, 143-160. GLOOR, U. ( 1970). Absorption, distribution and excretion of vitamin E and related substances. In International S~pos~um on Vitamin E, Hakone, pp. 3-15 Kyoritsu Shippan Co., Ltd., Tokyo, 1972. state
LIVER GREEN, and rat. ANN,
MITOCHONDRIA
AND
VITAMIN
E
243
J., DIPLOCK, A. T., BUNYAN, J., MCHALE, D., and MUTW, I. R. (1967). Vitamin E 1. Dietary unsaturated fatty acid stress and the metabolism of tocopherol in the Brit. J. Nutr. 21, 69-102. F. A., ad THOMPSON, T. E. (1969). Synthetic lipid bilayer membranes. Ann. Rev. S~ESS.
Biochem.
38, 241-259.
KWGENBERG, M. ( 1970). Functional and topochemical organisation of the respiratory chain. In Proceedings of the Eighth Congress of Biochemistry, Switzerland, p. 154. LEHNINGER, A. L. (1970). Biochemistry. The Molecular Basis of Cell Structure and Function. Worth, New York. LEONARD, P. J., and LOSOWSKY, M. S. (1967). Relationship between plasma vitamin E level and peroxide hemolysis test in human subjects. Amer. ]. Clin. Nutr. 20, 795-798. LUCY, J. A. ( 1972). Functional and structural aspects of biological membranes: A suggested structural role for vitamin E in the control of membrane permeability and stability. Ann. N.Y. Acad. Sci. 203, 411. MOLENAAR, I., Vos, ‘J., JAGER, F. C., and HOMMES, F. A. (1970). The effect of vitamin E deficiency on the ultrastructure of intestinal epithelial cells and their membranes in particular. In, International Symposium on Vitamin E, Hakone, pp. 76-101 Kyoritsu Shuppan, Co., Ltd., Tokyo, 1972. REITH, A., BRDICZKA, D., NOLTE, J., and STAUDTE, H. W. ( 1973). The inner membrane of mitochondria under influence of triiodothyronine and riboflavin deficiency in rat heart muscle and liver. Erptl. CeU Res. 77, I-14. RIEDE, U. N., STITNY, C., ALTHAUS, S., und Roun, H. P. (1972a). Ultrastrukturell-morphometrische Untersuchungen der Rattenparenchymzelle nach chronischer Vitamin E-Mangeldi%. Beitr. Path. 145, 24-36. RIEDE, U. N., SENN, E., und ROHR, H. P. ( 197213). Morphometrische Untersuchung der Lebermitochondrien beim Vitamin E-Mange1 der Ratte. Cytobiobgie 5, 181-189. ROIIR, H. P., OBPRIIOLZER, M., BARTSCH, G., and KELLER, M. (1976). Morphomctry in expcrimental pathology (methods, baseline data and applications). ht. Rev. Erp. Pathol. 15, 233-326. SCHWARZ, K. ( 1972). The cellular mechanism of vitamin E action: Direct and indirect effects of a-tocopherol on mitochondrial respiration, Ann. N.Y. Aced. Sci. 203, 45-52. SCHU~IETER, U., TAMM, R., WEISER, H., und WISS, 0. ( 1966). Zur Synthese und Vitamin-EWirksamkeit von Tocopheraminen und ihren N-Alkyl-Derivaten. Helv. Chim. Acta 49,
2297-2302. TAPPEL, A. G. (1962). Vitamin E as the biological lipid antioxidant. In Harris, R. S., and Hormones, Advances in Research Wool, I. G., Marrian, G. F., Thimann, K., “Vitamins and Applications.” Vol. 20, 493-509 Academic Press, New York-London. Vos, J., MOLENAAR, I., SEARLE VAN LEEUWEN, M., and HOMMES, F. A. (1972). Mitochondrial and microsomal membranes from livers of vitamin E-deficient ducklings. Ann. N.Y. Acad. Sci. 203, 74-80. WEIBEL, E. R. ( 1969). Stereological principles for morphometry in electron microscopic
cytology. ht. Rev. Cytol. 26, 235-302. WEIBEL, E. R. (1973). Stereological techniques for electron microscopic morphometry. In Hayat, M. A., Principles and Techniques of Electron Microscopy: Biological Applications, Vol. 3, 237-296 Van Nostrand Reinhold, New York . WEISER, H. ( 1969). Die Wirkung des Vitamins E. auf den Hamolysevorgang In Kress, H. Frhr. von, und Blum, K. U., Vitamine A, E und K, klinische und physiologisch-chemische Probleme, pp. 309-319 F. K. Schattauer Verlag, Stuttgart-New York. Wrss, O., BUNNELL, R. H., and GLOOR, U. ( 1962). Absorption and distribution of vitamin E in the tissues, Vitamins and Hormones 20, 441-455. YEH, Y. Y., and JOHNSON, R. M. ( 1973). Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline. Arch. Biochem. Biophys. 159, 821-831.
Ultrastructural
AND
MOLECULAR
PATHOLOCY
24, 236-243
and Stereological Study on Liver Mitochondrial M.
Received
May
(1976)
on the Effect Membranes
of Vitamin
E
FRIGC 1 AND H. P. ROHR 2
13, 1975,
and in revised
form
October
1, 1975
The quantitative composition of hepatocytes in mice was analyzed stereologically in vitamin E deficiency, after vitamin E supplementation and feeding of a standard diet. No difference between the three groups,of five animalseach,could be observed descriptively. Stereologically, the vitamin E deficiency group showed a significant increase of the surface density of the inner nlitochondria~ membrane. A model-like calculation of the “center to center distance” of cytochrome molecules revealed mean distances of 180-230 d in the control group, whereas, in the vitamin E deficiency group, these distances were nearly doubled.
1. INTRODUCTION The function of vitamin E as a biological antioxidant has been discussed from various points of view (Tappel, 1962; Henn and Thompson, 1969; Lucy, 1972). The antioxidant hypothesis has been confirmed by the possibility of repIacing vitamin E for certain functions, in vitro and in Vito, by synthetic antioxidants. Other studies (Green et at. 1967; Diplock et al. 1968, 1971) showed that the prevention of autoxidation of lipids may not be a direct function of vitamin E. Caygill et aZ. (1971) suggested a protective action of vitamin E for reduced selenium, The concentration and the intracellular distribution of selenide, which like vitamin E (Gloor, 1970; Wiss et al. 1962) is mainly found in mitochondria (Caygill et al. 1971) are strongly influenced by vitamin E. Lucy (1972) suggestsstabilization of membranes by vitamin E as an explanation for the increased vitamin E requirement where there is a high content of unsaturated fatty acids in the food. This stabilization is apparently caused by a physicochemical interaction between the phytyl side chain of vitamin E with polyunsatmated acyl chains of fatty acids. The stabilizing effect of vitamin E on the erythrocyte membrane was proved by Friedman et al. (1958) and Weiser (1969) by means of the hemolysis test. Molenaar et al. (1970) and Vos et al. (1972) observed, in ducklings and human biopsies, alterations in the electron microscopical contrast of mitochondria1 membranes from the epithelial cells of the jejunum, which were dependent on vitamin E. Riede et al. (1972a, b) observed in the hepatocytes of rats after chronic vitamin E deficiency an increase of the surface of the inner mitochondrial membrane by using ultrast~c~ral stereology. The occurrence of secondary symptoms in advanced vitamin E deficiency cannot be excluded. In the
Copyright ~11 rights
Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
LIVER
MITOCHONDRIA
AND VITAMIN
E
237
present study, an attempt was therefore made to determine quantitatively primary ultrastructural alterations of the mouse liver in vitamin E deficiency by assessing the vitamin E deficiency state (hemolysis test). 2. MATERIALS Experimental
AND
METHODS
Design
Male hybrid mice C57BLJ//CBA/J with an initial weight of 20-24 gm were divided into three groups of ten animals each: group 1 (ED) was fed a vitamin E-deficient ration ( Schwieter et al. 1966), group 2 (ES) was fed the same ration supplemented with 100 IU dLa-tocopherol acetate and group 3 (CD) obtained a commercial standard diet (NAFAG 869). A modified hemolysis test (Friedman et al. 1958; Leonard and Losowsky, 1967; Weiser, 1969), with a dialuric acid concentration of 4 mg/liter, was used to characterize the vitamin E deficiency. After a trial period of 17 weeks, tissue samples from the left liver lobule were taken under anaesthesia for stereological examination. For electron microscopy and the stereological model we refer to Rohr et al. ( 1976) and Weibel ( 1969, 1973). 3. RESULTS Hemolysis Test and Deficiency Symptoms
Table I shows the values from the hemolysis test with two different dialuric acid concentrations. In all tests, the animals from the vitamin E-deficient group showed a high hemolysis rate ( >92% ), which was significantly different from that of groups 2 and 3 (P < 0.05). With 16 mg dialuric acid/liter, the hemolysis rate is high also for groups 2 and 3 (71.5%, 70.4%, group 1 97.4%), but there is, however, no difference between groups 2 and 3. The low dialuric acid concentration of 4 mg/l results in a significant difference between groups 2 and 3 (P < 0.05). TABLE Hemolysis
Test in Animals of Group Vitamin E), and Group
Dialuric concentration Hemolysis
Mean Standard
1 (Vitamin E Deficiency), Group 2 (Supplemented 3 (CD) a Week Prior to Conclusion of Trial
Vitamin E deficiency
Group acid mg/l rate
error
in
70
(SM)
I
Vitamin E supplementation
different different
from from
CD
4
16
4
16
4
16
94.6 92.6 96.9 98.6 98.6
98.5 98.6 92.3 98.7 98.8
31.8 40.2 38.5 34.9 41.4
64.6 61.3 81.5 81.0 68.9
26.5 20.0 34.7 22.9 29.2
74.6 59.7 81.5 74.6 61.5
96.3” 1.17
97.4” 1.27
37.4* 1.76
71.5 4.17
26.7 2.54
70.4 4.19 __-
0 Significantly b Significantly
with
groups group
2 and 3, P < 0.05. 3, P < 0.05.
number
volume surface volume surface
Mitochondria
RERb RERb SERc SERC
cma/cnlJ m2/cm3 em3/cm3 ma/cm3
cms3
cmS/cm3 om8/cmz ema/cm3 cm3/cm3 mz/cm3 m*/cm3 cm-a
Unit
0 = Shape factor. a Significance in the l-test: 1, comparison P 6 0.05 at 8 degrees of freedom. b RER = Rough endoplaamic retkulum. * SER = Smooth endoplasmic reticulum.
volume volume volume volume surface surface number
Paramet~er
Hepatocyte Cell nucleus Cytoplasma ~~itoehondria Xlitochondria Mitochondria Xitochondria
___-.
Compart,ment
vitamin
‘5. t I 0; 0; 2;3; 0;
VW vvx &MC NW
0;
VVSEX SWEi
E supplementation/CD;
1; 0;
&RER
0;
V VREK
(@ = 1.4.;) NVM (j3 = 2.25) 0;
0;
&MO
3;
VVNH
Significancea
Values
VVR
Symbol
Stereological
0.3284 1.4214 0.0136 0.1908
Volume
Liver
E supplementation/vitamin
0.0258 0.133 0.0030 0.0432
0.0062
0.0084 0.0250 0.0091 0.0073 0.0227 0.0701 0.0102
SE
E deficiency
* 1012
0.0774.10’2
0.9176 0.0840 0.8336 0.1646 0.4674 1.3184 0.1292
?U
Vitamin
to the Unit
II
2, vitamin
Related
TABLE
0.302S 1.7328 0.0140 0.1464
0.0794
0.0543 0.1945 0.0033 0.0334
0.0064
0.0122 0.0139 0.0131 0.006 0.0667 0.1063 0.0105
SE
IlZ deficiency;
* 10’2
0.9010 o.osso 0.8130 0.1662 0.4426 0.9894 0.1320.10’2
m
Vitamin E supplementation
3, CD/vitamin
0.2834 1.1764 0.0174 0.3316
0.0894
0.0248 0.0740 0.0035 0.0887
0.0077
0.0108 0.0139 0.0092 0.0088 0.0256 0.0832 0.0128
SE
E deficiency;
* 10’2
0.8812 0.0620 0.8192 O.i666 0.3366 0.7726 0.1488~10’~
‘VL
CD
il
z:
G u
3 $j
LIVER
M~OCHONDRIA
AND ~TAMIN
TABLE Stereological Compartment
Parameter
E
239
III
Values Related to Unit Volume Cytoplasma of Hepatocytes Unit
Symbol
SiinifiC*nCe~
Vitamin defi&noy
E
Vitamin E supplementation m
CD m
SE
m
SE
SE
0.1906 0.5412
0.0095 0.0306
0.1940 0.5120
0.0079 0.0724
0.1992 0.4034
0.0088 0.0305
Mitochondrium
volume surface
Crn~/Ornf rn~/Crn~
Z$20
0:
gi&hondnum Mitochondr+n
surface vollmle
cm*/cm* m~/cm*
VVRER/VVC SYMC/ VW
z i3 ;
1.5260 0.3790
0.0290 0.0862
0.3532 1.1478
0.0615 0.109%
0.3390 0.9264
0.0287 0.1ooe
EiY SER:
surface volume surface
m~/mn* cm*/cm* m*/cmE
SVR&R/VVO vv8ER/vvo SVE~/V~O
of yj 0;
0.0160 1.6494 0.2214
0.1676 0.0035 0.0512
0.0166 2.0110 0.1698
0.2033 0.0037 0.037
0.02m 1.4092 0.3992
0.0873 0.0043 0.1096
5 Siiificanca in vitmnin E defkienoy, b RER = Rough c SER = Smooth
Student’s test: 1. comparison 3. CD/vitamin E deficiency, endoplasmio reticulum. endoplaamio reticulum.
vitamin
No difference in weight development served between the groups. Ultrastructural
E suppleme~~tion/CD, at 8 degrees of freedom.
P < 0.05
or behavior
2. vitamin
E s~ppiementation/
of the animals could be ob-
Aesdts
No differences between the three trial groups can be evaluated descriptively. The mitochondria are built up normally and the efectron density of the matrix is unchanged. Stereological
Results
The volume density of the mitochondria in the reference systems liver, hepatocyte and cytoplasm, does not show any significant difference between the three trial groups, control group (CD), vitamin E-supplemented group (ES), and vitamin E-deficient group ( ED). The main finding consists of ‘an increase of the surface density of the inner membrane of the mitochondria in the vitamin E-deficient group (Tables II and III). The surface density of the outer membrane of the mitochondria is only increased signi~cantly when compared with the CD group (Tables III and IV). Also the group supplemented with vitamin E showed an increased surface density of the inner mitochondrial membrane in comparison to the CD controls. When related to the unit volume mitochondria, these values are also greater in the deficient and supplemented groups compared to the CD group (Table IV). The volume density of the mitochondria (Tables II and III) as well as the mean single volume of mitochondria (Table IV), however, remain unchanged.
Stereological ParWleter
Mitoohondria: Outer membrane Outer membrane Inner membrane Inner membrane Mean volume (@ = 1.45)
:;;z~ surface surface volume
Tinit
m~/cm~ WQ m~/om: em’ tm0
n Significance in Student’s &teat: vibmin E deficiency; 3. CD/vitamin
Symbol
SVMO/VVM pMM/;~ v f$y/gNvy
1. clomp&son E deficiency;
TABLE IV Values of Mitwhondria Significnnce’
3; 3; 2;3; 1;2;3; 0;
vitamin
P 6 0.05
Vitamin deficiency
E
m
SE
2.6638 3.7544 8.0118 10.3816 1.3016
0.2086 0.4768 0.3419 0.3508 0.1128
E suppbmentation/C.D. at 8 degrees Of freedom.
Vitamin E supplementation m
2.6654 4.1022 6.9664 7.4934 1.2662
SE
0.3897 1.0630 0.6511 0.6221 0.1066
2. vitamin
CD
~ pn
2.0360 2.3404 4.6714 5.2394 1.1718
E supplementation/
SE
0.1911 0.2833 0.6097 0.4574 0.1639
FRIGG AND ROHR
240
TABLE II~~ueIl~
of a-Tocopherol on ~fitoehondrial Cytochromes (according
&011p
mg Mitochondrial protein/gm liver .-_-.--___-
__..-Standard Vitamin Vitamin a Data
diet (CD.) E supplementation E deficiency from
Table
V Proteins to Schwarq nMol
14.99 9.06 8.31
and on Various 1972)
Cytoohrome/mg chondrial protein
of Their
mito-
a
b
Cl
c
0.26 0.31 0.2.i
0.35 . 0.37 0.31
0.22 0.27 0.22
0.2.3 0.2.: 0.27
SYd~VC” (m”/cm3)
0.9264 1.1478 I.5260
III.
The volume and surface values of the rough and smooth elldopIasnlic reticulum show no changes in all three trial groups. 4. DISCUSSION The surface density of the mitochondrial inner membrane of the vitamin Edeficient group is increased significantly (Fig. 1) compared to the two control groups. The surface to volume ratio of the inner membrane of the mitochondria (Sv&Vvn) is higher in the vitamin E-deficient group than in the control group (Fig. 1). A swelling of the mitochondria with chronic vitamin E deficiency, which should result stereologically in a reduction of this ratio can, therefore, be excluded. Rather can an augme~~tation of the inner nlitochondrial membrane be assumed, since the volume density of the mito~hondria remains constant in all reference systems. A genuine enlargement of the surface of the mitochondrial membranes requires, on the one hand, the formation of membrane elements, as well as, on the other hand, the synthesis of membrane-bound enzymes. Membrane elements and %MC1UUM m2h3
T
ED
ES
1. Surface density of mitochondrial outer to the volume density of mitochondria (VW). FIG.
CD
ED
( SvMo)
ES CD
and
inner
( SvMa)
membranes
related
LIVER
MITOCHONDRIA
AND VITAMIN
TABLE
E
241
VI
Mean Distances (A) of Cytochrome Molecules in Animals with a Normal Diet (CD), in Animals Fed a Vitamin E-Deficient Diet with and without Vitamin E Supplementationa Group
Normal diet (C.D.) Vitamin
E supplementation
Vitamin
E deficiency
a The cytochrome concentrations
Cytochrome
A % A % A %
a
b
Cl
c
210.0 100.0 280.0 130.0 380.0 180.0
180.0 100.0 260.0 140.0 340.0 180.0
230.0 100.0 300.0 130.0 400.0 170.0
220.0 100.0 310.0 140.0 360.0 170.0
according to Schwarz (1972) were used for the calculations.
mitochondrial enzymes are mainly synthesized in the rough endoplasmic reticulum and, to a smaller extent (approximately 10% ), intramitochondrially ( Beattie, 1971). Under pathological conditions, however, it is conceivable that a distinction must be made between various proliferation types of mitochondrial membranes: 1. Membrane-bound enzymes and membrane elements are both formed proportionally to an increased extent. 2. Only membranes are synthesized. The formation of enzymes remains constant or is reduced. In such a case, the occurrence of membranes with a “diluted” enzyme content must be assumed. 3. The membrane formation is enhanced, the enzyme formation, however, more intensively in comparison to the membrane formation. There result membranes with a compacter enzyme content. Neither descriptively, stereologically, nor biochemically can a distinction be made between these three hypothetical types of membrane proliferation. Only a suitable correlation with stereological and biochemical data can furnish further elucidation (cf. Reith et al. 1973). Therefore, it was tried to confirm this evidence of a disturbed membrane integrity by means of a model-like calculation of the mean enzyme distances (center to center distance) on the mitochondrial inner membrane. According to Schwarz (1972), in vitamin E deficiency, the content of mitochondrial protein per gm liver tissue and cytochrome concentrations per mg mitochondrial protein are reduced (Table V). Since the arrangement of the groups in the studies of Schwarz (1972) corresponds to a great extent with the trial plan described here, the results of Schwarz (1972) were used to calculate the mean enzyme distances. For the calculation of the enzyme distances, we made the following assumptions (cf. Reith et al. 1973, and Rohr et al. 1976) : 1. The enzymes are distributed homogeneously in the inner membrane. 2. The density of the enzyme content should be expressed as mean area which may be assigned to one enzyme, respectively, as “center to center distance.”
242
FRIGG
AND
ROHR
The calculation of this mean “center to center distance” is explained by the following example (CD control group) : Cytochrome a : 0.26 n&fof/mg mitochondrial protein Mitochondrial protein: 14.99 mg/gm liver tissue Surface of the inner membrane 0.926 m2/cm3 liver tissue ( &MC/VVC ) : Cytochrome share in Iiver tissue: 3.898 nA4oI,/gm liver, corresponding to 23.478- 1Ol4 molecules Mean area (F) per molecule: 39.441 A Mean distance (d) of cytochrome a molecule assuming a hexagonal arrangement: d = 213 A It appeared that, in the CD control group, the mean distances of the cytochrome molecules lie in the range of 180-230 A. Reith et aE. (1973) calculate values of 260 A, whereas, according to Lehninger ( 1970)) the “center to center distances” of the respiratory units should amount to 230 A. In the heart muscle, the mean statistical distance of the cytochrome a molecule is 220 A (Klingenberg,
1970). Chronic vitamin E deficiency Ieads to nearly double the mean distances of the four cytochromes (Table VI). The vitamin E-supplemented diet, too, leads to an increase of 30-40s of these enzyme distances. These correlated biochemicostereological rest&s show that the formation of mitochondrial inner membrane is enhanced in vitamin E deficiency, whose cytochrome density, however, is reduced. These findings indicate that the formation of mitochondrial inner membrane increases in vitamin E deficiency states, the synthesis of the membranebound cytochrome, however, does not seem to be enhanced proportionaIIy. The stereologica1 findings as well as the model-like calculations of the mean “center to center distances” of the cytochrome molecules show that, in a chronic vitamin E deficiency state, structurally and thus functionally modified (defective) membranes are formed to a greater extent. Thus, the investigations of Schwarz (1972), Yeh and Johnson ( 1973) as well as Carabello (1974), which point to a disturbed membrane integrity with vitamin E deficiency, are further supported. REFERENCES BEATTIE, D. (kiABELL0, Biochem. CAYGILL, C. intracellular
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