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The effect of vitamin E deficiency on the oxidation of tricarboxylic acid cycle intermediates
The Effect of Vitamin E Deficiency on the Oxidation of Tricarboxylic Acid Cycle Intermediates’s 2 I. M. Weinstock,
I. Shoichet, A. D. Goldrich
the Departments of Psychiatry College, the Russell Sage Institute
From
New Received
and
Medicine,
of Pathology, York,
New
February
and
and A. T. Milhorat Cornell University Medical The New York Hospital,
York 18, 1955
Muscular dystrophy resulting from vitamin E deficiency is associated with increased oxygen consumption by skeletal muscle strips and slices (l-6). Recently it was reported that during deficiency of this vitamin, endogenous respiration and xanthine oxidase activity of whole homogenates of liver (7, 8) and endogenous respiration of liver slices (9) are also increased. This report is concerned with the effect of vitamin E deficiency on the oxidation of tricarboxylic acid cycle intermediates by washed particulate preparations of rabbit liver. EXPERIMENTAL Young rabbits (550-850 g.) were maintained on the vitamin E-deficient diet of Goettsch and Pappenheimer (10) treated with 1% FeCla Symptoms of the stage II (11) state of the deficiency (loss of appetite and weight, and urinary creatinecreatinine ratios greater than 1.0) became evident in 4-5 weeks. Control animals received the untreated Goettsch and Pappenheimer diet supplemented with 25 mg. y. dZ-o-tocopherol.3 Animals were sacrificed, after stunning, by exsanguina1 Aided by grants from Muscular Dystrophy Associations of America, Inc., and the National Institutes of Health, U. S. Public Health Service. 2 Preliminary reports of some of these data were presented before the Federation of American Societies for Experimental Biology, April, 1954, and the Third Medical Conference of Muscular Dystrophy Associations of America, Inc., October, 1954. 8 Some control animals received the FeCl&reated Goettsch and Pappenheimer diet supplemented with tocopherol. Results with these animals were the same as those obtained with the other controls and are included in the averaged control values.
EFFECT OF VITAMIN
E DEFICIENCY
497
tion. Portions of the liver were homogenized in buffer of the following composition: 0.0128 M Na2HPOn, 0.123 M SaCl, 0.005 iV KCl, and 0.0012 M MgSOa , pH 7.7-7.8. The homogenate was strained through three layers of gauze, diluted with the buffer to 9-10 times the volume of the original portion of liver, and the particulate matter was separated by centrifugation at 6OQ X y for 10 min. The particulate matter residue was washed twice by resuspension in the buffer and b> centrifugation. The final residue was suspended in the buffer and diluted to a final volume 3-4 times that of the original portions of liver. All procedures were carried out at 0-3°C. Nitrogen determinations were routinely carried out on the final preparation of particulate matter by nesslerization. One-milliliter aliquots of the suspension were added to Warburg flasks containing 10 J.&’ Mg++, 0.036-0.044 pM cytochrome c, 14 MMphosphate buffer pH 7.4-7.6, 3.6-4.4J~1 adenine nucleotide, and substrate. In experiments in which fluoride was used, the fluoride solution was added immediately after the enzyme (12) The final total volume of the incubation mixture was 2 ml. Center wells contained 0.2 ml. of 20% KOH and folded filter paper. Incubations were carried out at 3O”C., in air. Oxygen consumption was measured after a 7.5-10.min. equilibration period with the enzyme in the presence of the substrate, and values were extrapolated back to zero time and corrected for endogenous respiration. RESULTS
When incubat’ion mixtures were supplemented with adenosine triphosphate (ATP),4 enzyme preparations from vitamin E-deficient animals utilized 60400% more oxygen than did controls when oxidizing five of the six added substrates (Table I). With citrate as the added substrate, the slightly elevated oxygen consumption of the deficient preparations was not significant at less than the 5 % level. The increment in oxygen consumption depended on the severity of the deficiency. During early stages of vitamin E deficiency, oxygen consumption was only slightly higher than, or practically the same as, controls. The averages in Table I include data from animals in stage II as well as earlier states of vitamin E deficiency. Oxidation by both deficient and control preparations varied slightly with the protein nitrogen content of the washed homogenates. However, the large differences in oxygen consumption reported here could not be accounted for by differences in the amount of protein nitrogen within the range of concentrations used. Some of the washed homogenates were resuspended in 0.25 M sucrose and fractionated by the method of Schneider (13). The particulate fractions obtained 4 The ATP consisted of a number of different commercial preparations of “amorphous” or “crystalline” chromatographically purified ATP. The same preparation was used for each experiment and its control.
498
-
-
I ;
-
-
-
>
-
-
EFFECT oATPa
OF
VITAMIN
E
DEFICIENCY
+AMP’
499
ONone.
HOURS
1. Effect of adenine nucleotide supplementation on tricarboxylic acid cycle oxidation during vitamin E deficiency. Conditions as described in text. Results are averages of typical experiments with 5 pM of substrate. Deficient animals were in stage II of vitamin E deficiency. Some experimental points have been omitted for clarity. Control: 0 ATP, A ADP, + AMP, 0 none. Deficient: l ATP, A ADP, X AMP, n none. FIG.
by centrifugation at 600 and 8500 X g accounted for 80% (67-88 YG) and 20% (l&33%), respectively, of the nitrogen of t,he original salinewashed preparations.6 In a few experiments, saline-washed homogenates prepared by centrifugation at 1400 X g were studied. These homogenaks contain appreciable amounts of mitochondria (15, 16) and gave results that were qualit’atively similar to those obtained with material prepared at, BOO X g. s Portions of liver from control and deficient animals were homogenized in 0.25 JI sucrose-0.00018 M CaClz and strained through gauze. Nuclei (plus any unruptured cells) were prepared by the method of Hogeboom et al. (14). The final preparation was washed in 0.25 M sucroseeO.001 M Versene to remove Ca++. The IOR level of orygen consumption I))- such preparations was similar for both control and deficient groups when supplemented with ATP or ADP (unpublished results).
500
WEINSTOCK,
SHOICHET,
GOLDRICH
AND
MILHORAT
If the ATP in the incubation mixtures was replaced by an equal amount of ADP, oxygen consumption of the controls was increased to the levels of ATP-supplemented preparations from animals in stage II of deficiency (Fig. 1). The adenosine diphosphate (ADP) substitution had no marked effect on oxidation by “stage II deficiency” preparations, although it did increase oxygen consumption by liver preparations obtained from rabbits during the earlier stages of vitamin E deficiency. When citrate was the added substrate, oxygen consumption was essentially the same for both ATP- and ADP-supplemented control and deficient systems. The difference in oxygen consumption values between ATP-supplemented control and deficient preparations was due primarily to a more rapid decrease in the rate of oxidation by the control group. Initial rates of oxidation were essentially similar, as is most readily seen when cY-ketoglutarate was the substrate added. Oxidation by preparations e--o
r
I5 E,
CITRATE
ATP P-7
A--AADP
-ATP
o!KETOGLUTARATE
+ F
-ADP c
+ F FUN IARATE
18 15-
I
r’
-o-o-o-
HOURS
2. Effect of fluoride on tricarboxylic acid cycle oxidation during vitamin E deficiency. Conditions as described in text. Results are averages of typical experiments with 5 FM of substrate. Deficient animals were in stage II of vitamin E deficiency. Some experimental points have been omitted for clarity. l ATP, A ADP, 0 ATP + 0.015 M NaF, A ADP + 0.015 M NaF. FIG.
EFFECT
OF
VITAMIW
E
501
DEFICIESCY
supplemented with adenosine monophosphate (AMP) was similar to that of ADP-supplemented systems (Fig. 1) and there was no difference between the control and E-deficient groups. Failure to add any type of adenine nucleotide also eliminated the difference between control and E-deficient preparations, when a-ketoglutarate was the added sub&rate, and reduced oxygen consumption below the levels of ATP-supplemented controls with this substrate (Fig. 1). The addition of fluoride, to a final concentration of 1.5 X 1R2 31 (Fig. 2), did not reduce the oxygen consumption of ATP-supplement,ed deficient preparations to the levels of ATP-supplemented cont8rols, but did, to some extent, reduce the rate of oxidation in all systems. Higher concentrations of fluoride (0.045 M) also failed to reduce the oxygen consumption of ATP-supplemented deficient preparations to control levels. However, in ATP-supplemented control systems (except with citCONTROL
VITAMIN
E-DEFICIENT
l3I.2
l2II -
3 435.6
0
I
2
3
4
0 HOURS
I
2
3
4
FIG. 3. Tricarboxylic acid cycle oxidation and vitamin E deficiency. Effect of AT!? addition during the course of incubation. A : 4 rM ADP 0 min. B: 4 pM ADP Omin.+4pMATP60min.C:4~MADPOmin.+4~ctMATP30min.+3pM ATP 60 min. D: 4 pM ADP 0 min. + 4 pM ATP 30 min. -+- 3 pM ATP 90 min. E: 4 NM ADP 0 min. -+ 4 pM ATP 30 min. + 3 pM ADP 180 min. F: 4 pM ADP + 4 pM ATP 0 min. G: 4 pM ATP 0 min. + 4 pM A4TP 60 min. H: 4 phi! ATP 0 min.1:4pMADPOmin.2:4~MATPOmin.3:4~MATPOmin.+4~MATP 60 min. 4: 4pM ATP 0 min. 4 4pM ATP 30 min. f 3 pM ATP 60 min. 5: 4 ,I& ATPOmin.+4~MATP30min.+3~MATP90min.6:4~MADPOmin.+4 &M ATP 30 min. + 3 MM ATP 90 min. Other conditions as described in text.
502
WEINSTOCK,
SHOICHET,
GOLDRICH
AND
MILHORAT
rate as the added substrate), the addition of fluoride prolonged the period of oxygen consumption, so that the total amount of oxygen utilized was increased to the levels of the deficient state (Fig. 2). The effect of fluoride in increasing total oxygen consumption, while decreasing the rate of oxidation, has been reported by Harmon and Osborne (17) for muscle systems oxidizing a-ketoglutarate. Harmon and co-workers (17, 18) believe that fluoride, by maintaining ATP levels, helps to preserve the structural and functional integrity of mitochondria. The results of a number of experiments in which ATP was added during the course of incubation are reported in Fig. 3. The addition of ATP to ADP-supplemented control preparations caused a rapid decrease in oxidation rate and a total cessation of oxygen consumption within 1 hr. (curves B-E). Addition of ATP to ADP- or ATP-supplemented vitamin E-deficient preparations, stopped oxidation only after 2-2x hr. (curves 3-6) and did not lower oxygen consumption to the levels of corresponding controls (curves B-E). The inhibition of oxidation, which depended on the time of ATP addition (curves B-E and 3-6), was not affected by further supplementation with ATP (curves C-D and 4-6), and could not be reversed by subsequent addition of ADP (curve E). Addition of ADP at zero time (curve F), or further ATP supplementation at 1 hr. (curve G), did not affect oxidation by ATP-supplemented control preparations (curve H). DISCUSSION
The increased oxygen consumption by ATP-supplemented washed liver homogenates, during vitamin E deficiency, appeared after the earliest stages of muscular dystrophy, as indicated by increasing urinary creatine excretion and decrease in weight gain by the rabbit. The very large increments in oxygen consumption appeared during stage II (11) of the deficiency. Because of this time factor, the increased oxidation of tricarboxylic acid cycle intermediates, like the increased liver xanthine oxidase activity (7), may be secondary to some more fundamental effect of the deficiency. We have no explanation for the unique behavior of added citrate, as compared to the other substrates investigated, except to note the report of Christie and Judah (19) that the oxidation of added citrate may differ from the oxidation of citrate formed intramitochrondrially. Although substitution of ADP for ATP increased the oxygen consumption of control liver preparations to that of the deficient group, the
EFFECT
OF
VITAMIN
E DEFICIENCY
503
present evidence does not indicate that the difference observed with ATP-supplemented preparations was due to a greater availability of high-energy phosphate-bond acceptor systems during vitamin E deficiency. On the basis of reports concerning the myokinase and adenosinetriphosphatase (ATPase) activity of various liver preparations, “inhibition” of oxygen consumption due to saturation of high-energy phosphate-bond acceptor systems, or decrease of inorganic phosphate in the incubation mixtures, would not be expected with the type of preparations used in these studies (20-28). Morgulis and Jacobi (29) at one time did suggest that the increased respiration of skeletal muscle during vitamin E deficiency might be due to increased ATPase activity. However they (30) and other investigators (31-32) were unable to detect any increase in ATPase activity during the deficiency. Vitamin E deficiency also had no effect on ATPase activity of liver preparations under conditions similar to those employed in the studies reported here (33). In the investigations being reported, the addition of fluoride to a final concentration of 1.5 X 1W2 M, which should have an appreciable effect on ,4TPase and myokinase activity (12, 20, 21, 27, 34, 35), did not markedly reduce the total oxygen consumption by ATP-supplemented deficient liver preparations to control levels. It was also found that oxygen consumption by both control and vitamin E-deficient liver preparations supplemented with AMP was at the same high level as ADP-supplemented systems. Oxygen consumption by controls, then, does not appear to be limited by myokinase activity which is probably unaffected by the deficiency. Finally, supplementation of control preparations with a mixture of ATP and ADP (Fig. 3, F) did not increase oxygen consumption over controls containing only ATP (Fig. 3, H). This was contrary to the result that might be expected if the increase in oxygen consumption obtained by substituting ADP for ATP was due to the ability of ADP to serve as a high-energy phosphate-bond acceptor system. The difference in oxygen consumption by washed liver homogenates during vitamin E deficiency depended on the presence of ATP in the incubation system. Addition of ATP during the incubation resulted in a rapid decrease and complete cessation of oxygen consumption by ADPsupplemented control preparations (Fig. 3, B-E), which could not be reversed by subsequent addition of ADP (Fig. 3, E). Addition of ATP during the incubation of vitamin E-deficient preparations had a much smaller inhibitory effect (Fig. 3, 3-6) and oxygen consumption was not reduced to the levels of corresponding controls (Fig. 3, B-E). The finding
504
WEINSTOCK,
SHOICHET,
GOLDRICH
AND
MILHORAT
that addition of ATP during the incubation of control preparations did not increase oxygen consumption and, in fact, caused its cessation, indicated that the difference in oxidation was not due to a more rapid destruction of ATP. These results suggest that during vitamin E deficiency there is a defect in a mechanism controlling oxidation by washed liver homogenates. Such a control mechanism appears to be dependent on the presence of ATP in the incubation system and independent of the level of highenergy phosphate-bond acceptor systems, at least as represented by ADP.6 A mechanism controlling oxidation, other than by the level of phosphate-bond acceptor systems, has been reported by Johnson and Ackermann (36) and Christie and Judah (19). These investigators have found that the addition of nuclei stimulated oxidation by mitochondria by some means other than the effect of the nuclei on the levels of acceptor systems and to a greater extent than added hexokinase and glucose. Although there is no direct experimental evidence, it is tempting to speculate that vitamin E deficiency may affect yet another mechanism controlling oxidation that is independent of the levels of phosphate-bond acceptor systems. SUMMARY
Oxidation of five of six added tricarboxylic acid substrates by washed liver homogenates from vitamin E-deficient rabbits was 60-600 % higher than controls when supplemented with ATP. Fluoride did not reduce oxidation to control levels. Replacing ATP with ADP or AMP eliminated the difference in oxygen consumption. REFERENCES 1. VICTOR,
J., Am. J. Physiol. 106,229 (1934). MADSEN, L. L., J. Nutrition 11, 471 (1936). FRIEDMAN, I., AND MATILL, H. A., Am. J. Physiol. 131, 595 (1941). HOUCHIN, 0. B., AND MATILL, H. A., J. Biol. Chem. 146, 301 (1942). BASINSKI, D. H., AND HUMMEL, J. P., J. Biol. Chem. 167, 339 (1947). HIJMMEL, J. P., AND BASINSKI, D. H., J. BioZ. Chem. 172, 417 (1948). DINNING, J. S., J. BioZ. Chem. 202, 213 (1953). RICHERT, D. A., AND WESTERFELD, W. W., Proc. Sot. Exptl. Biol. Med. 468 (1953). 9. ROSENKRANTZ, H., J. BioZ. Chem. 214, 789 (1955).
2. 3. 4. 5. 6. 7. 8.
tions
6 In a few experiments the addition of hexokinase and glucose did not increase oxygen consumption (unpublished results).
to these
prepara-
84,
EFFECT
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
OF
VITAMIN
E
DEFICIENCY
505
GOETTSCH, M., ASD PAPPENHEIMER, A. M., J. Erpt2. &fed. 64, 145 (1931). MACKENZIE, C. G., AND MCCOLLUM, E. V., ,J. IY~ttrition 19, 345 (1940). POTTER, V. It., J. Biol. Chenz. 169, 17 (1947). ScHNEIr~ER, W. C., J. Biol. Chem. 176, 259 (1948). HOGEBOO~I, G. H., SCHNEIDER, W. C., AND STREIBICII, hI. .J., J. HioZ. Chem. 196, 111 (1952). SCHSEII~ER, W. C., J. Biol. Chem. 166, 585 (1946). HOGEBOOM, G. H., SCHNEIDER, W. C., AND PALLAUE, G. E., J. Biol. Chews. 172, 619 (1948). HARMOS, J. W., AND OSBORNE, U. H., J. Exptl. h!ied. 98, 81 (1953). HARMOS, J. W., AND FEIGELSON, M., Exptl. Cell Research 3, 509 (1952). CIIRISTIE, G. S., AND JUDAH, J. D., Proc. Roy. Sot. (London) B141,420 (1953). CROSS, R. J., TAGGART, J. V., Covo, G. A., .
I. Shoichet, A. D. Goldrich
the Departments of Psychiatry College, the Russell Sage Institute
From
New Received
and
Medicine,
of Pathology, York,
New
February
and
and A. T. Milhorat Cornell University Medical The New York Hospital,
York 18, 1955
Muscular dystrophy resulting from vitamin E deficiency is associated with increased oxygen consumption by skeletal muscle strips and slices (l-6). Recently it was reported that during deficiency of this vitamin, endogenous respiration and xanthine oxidase activity of whole homogenates of liver (7, 8) and endogenous respiration of liver slices (9) are also increased. This report is concerned with the effect of vitamin E deficiency on the oxidation of tricarboxylic acid cycle intermediates by washed particulate preparations of rabbit liver. EXPERIMENTAL Young rabbits (550-850 g.) were maintained on the vitamin E-deficient diet of Goettsch and Pappenheimer (10) treated with 1% FeCla Symptoms of the stage II (11) state of the deficiency (loss of appetite and weight, and urinary creatinecreatinine ratios greater than 1.0) became evident in 4-5 weeks. Control animals received the untreated Goettsch and Pappenheimer diet supplemented with 25 mg. y. dZ-o-tocopherol.3 Animals were sacrificed, after stunning, by exsanguina1 Aided by grants from Muscular Dystrophy Associations of America, Inc., and the National Institutes of Health, U. S. Public Health Service. 2 Preliminary reports of some of these data were presented before the Federation of American Societies for Experimental Biology, April, 1954, and the Third Medical Conference of Muscular Dystrophy Associations of America, Inc., October, 1954. 8 Some control animals received the FeCl&reated Goettsch and Pappenheimer diet supplemented with tocopherol. Results with these animals were the same as those obtained with the other controls and are included in the averaged control values.
EFFECT OF VITAMIN
E DEFICIENCY
497
tion. Portions of the liver were homogenized in buffer of the following composition: 0.0128 M Na2HPOn, 0.123 M SaCl, 0.005 iV KCl, and 0.0012 M MgSOa , pH 7.7-7.8. The homogenate was strained through three layers of gauze, diluted with the buffer to 9-10 times the volume of the original portion of liver, and the particulate matter was separated by centrifugation at 6OQ X y for 10 min. The particulate matter residue was washed twice by resuspension in the buffer and b> centrifugation. The final residue was suspended in the buffer and diluted to a final volume 3-4 times that of the original portions of liver. All procedures were carried out at 0-3°C. Nitrogen determinations were routinely carried out on the final preparation of particulate matter by nesslerization. One-milliliter aliquots of the suspension were added to Warburg flasks containing 10 J.&’ Mg++, 0.036-0.044 pM cytochrome c, 14 MMphosphate buffer pH 7.4-7.6, 3.6-4.4J~1 adenine nucleotide, and substrate. In experiments in which fluoride was used, the fluoride solution was added immediately after the enzyme (12) The final total volume of the incubation mixture was 2 ml. Center wells contained 0.2 ml. of 20% KOH and folded filter paper. Incubations were carried out at 3O”C., in air. Oxygen consumption was measured after a 7.5-10.min. equilibration period with the enzyme in the presence of the substrate, and values were extrapolated back to zero time and corrected for endogenous respiration. RESULTS
When incubat’ion mixtures were supplemented with adenosine triphosphate (ATP),4 enzyme preparations from vitamin E-deficient animals utilized 60400% more oxygen than did controls when oxidizing five of the six added substrates (Table I). With citrate as the added substrate, the slightly elevated oxygen consumption of the deficient preparations was not significant at less than the 5 % level. The increment in oxygen consumption depended on the severity of the deficiency. During early stages of vitamin E deficiency, oxygen consumption was only slightly higher than, or practically the same as, controls. The averages in Table I include data from animals in stage II as well as earlier states of vitamin E deficiency. Oxidation by both deficient and control preparations varied slightly with the protein nitrogen content of the washed homogenates. However, the large differences in oxygen consumption reported here could not be accounted for by differences in the amount of protein nitrogen within the range of concentrations used. Some of the washed homogenates were resuspended in 0.25 M sucrose and fractionated by the method of Schneider (13). The particulate fractions obtained 4 The ATP consisted of a number of different commercial preparations of “amorphous” or “crystalline” chromatographically purified ATP. The same preparation was used for each experiment and its control.
498
-
-
I ;
-
-
-
>
-
-
EFFECT oATPa
OF
VITAMIN
E
DEFICIENCY
+AMP’
499
ONone.
HOURS
1. Effect of adenine nucleotide supplementation on tricarboxylic acid cycle oxidation during vitamin E deficiency. Conditions as described in text. Results are averages of typical experiments with 5 pM of substrate. Deficient animals were in stage II of vitamin E deficiency. Some experimental points have been omitted for clarity. Control: 0 ATP, A ADP, + AMP, 0 none. Deficient: l ATP, A ADP, X AMP, n none. FIG.
by centrifugation at 600 and 8500 X g accounted for 80% (67-88 YG) and 20% (l&33%), respectively, of the nitrogen of t,he original salinewashed preparations.6 In a few experiments, saline-washed homogenates prepared by centrifugation at 1400 X g were studied. These homogenaks contain appreciable amounts of mitochondria (15, 16) and gave results that were qualit’atively similar to those obtained with material prepared at, BOO X g. s Portions of liver from control and deficient animals were homogenized in 0.25 JI sucrose-0.00018 M CaClz and strained through gauze. Nuclei (plus any unruptured cells) were prepared by the method of Hogeboom et al. (14). The final preparation was washed in 0.25 M sucroseeO.001 M Versene to remove Ca++. The IOR level of orygen consumption I))- such preparations was similar for both control and deficient groups when supplemented with ATP or ADP (unpublished results).
500
WEINSTOCK,
SHOICHET,
GOLDRICH
AND
MILHORAT
If the ATP in the incubation mixtures was replaced by an equal amount of ADP, oxygen consumption of the controls was increased to the levels of ATP-supplemented preparations from animals in stage II of deficiency (Fig. 1). The adenosine diphosphate (ADP) substitution had no marked effect on oxidation by “stage II deficiency” preparations, although it did increase oxygen consumption by liver preparations obtained from rabbits during the earlier stages of vitamin E deficiency. When citrate was the added substrate, oxygen consumption was essentially the same for both ATP- and ADP-supplemented control and deficient systems. The difference in oxygen consumption values between ATP-supplemented control and deficient preparations was due primarily to a more rapid decrease in the rate of oxidation by the control group. Initial rates of oxidation were essentially similar, as is most readily seen when cY-ketoglutarate was the substrate added. Oxidation by preparations e--o
r
I5 E,
CITRATE
ATP P-7
A--AADP
-ATP
o!KETOGLUTARATE
+ F
-ADP c
+ F FUN IARATE
18 15-
I
r’
-o-o-o-
HOURS
2. Effect of fluoride on tricarboxylic acid cycle oxidation during vitamin E deficiency. Conditions as described in text. Results are averages of typical experiments with 5 FM of substrate. Deficient animals were in stage II of vitamin E deficiency. Some experimental points have been omitted for clarity. l ATP, A ADP, 0 ATP + 0.015 M NaF, A ADP + 0.015 M NaF. FIG.
EFFECT
OF
VITAMIW
E
501
DEFICIESCY
supplemented with adenosine monophosphate (AMP) was similar to that of ADP-supplemented systems (Fig. 1) and there was no difference between the control and E-deficient groups. Failure to add any type of adenine nucleotide also eliminated the difference between control and E-deficient preparations, when a-ketoglutarate was the added sub&rate, and reduced oxygen consumption below the levels of ATP-supplemented controls with this substrate (Fig. 1). The addition of fluoride, to a final concentration of 1.5 X 1R2 31 (Fig. 2), did not reduce the oxygen consumption of ATP-supplement,ed deficient preparations to the levels of ATP-supplemented cont8rols, but did, to some extent, reduce the rate of oxidation in all systems. Higher concentrations of fluoride (0.045 M) also failed to reduce the oxygen consumption of ATP-supplemented deficient preparations to control levels. However, in ATP-supplemented control systems (except with citCONTROL
VITAMIN
E-DEFICIENT
l3I.2
l2II -
3 435.6
0
I
2
3
4
0 HOURS
I
2
3
4
FIG. 3. Tricarboxylic acid cycle oxidation and vitamin E deficiency. Effect of AT!? addition during the course of incubation. A : 4 rM ADP 0 min. B: 4 pM ADP Omin.+4pMATP60min.C:4~MADPOmin.+4~ctMATP30min.+3pM ATP 60 min. D: 4 pM ADP 0 min. + 4 pM ATP 30 min. -+- 3 pM ATP 90 min. E: 4 NM ADP 0 min. -+ 4 pM ATP 30 min. + 3 pM ADP 180 min. F: 4 pM ADP + 4 pM ATP 0 min. G: 4 pM ATP 0 min. + 4 pM A4TP 60 min. H: 4 phi! ATP 0 min.1:4pMADPOmin.2:4~MATPOmin.3:4~MATPOmin.+4~MATP 60 min. 4: 4pM ATP 0 min. 4 4pM ATP 30 min. f 3 pM ATP 60 min. 5: 4 ,I& ATPOmin.+4~MATP30min.+3~MATP90min.6:4~MADPOmin.+4 &M ATP 30 min. + 3 MM ATP 90 min. Other conditions as described in text.
502
WEINSTOCK,
SHOICHET,
GOLDRICH
AND
MILHORAT
rate as the added substrate), the addition of fluoride prolonged the period of oxygen consumption, so that the total amount of oxygen utilized was increased to the levels of the deficient state (Fig. 2). The effect of fluoride in increasing total oxygen consumption, while decreasing the rate of oxidation, has been reported by Harmon and Osborne (17) for muscle systems oxidizing a-ketoglutarate. Harmon and co-workers (17, 18) believe that fluoride, by maintaining ATP levels, helps to preserve the structural and functional integrity of mitochondria. The results of a number of experiments in which ATP was added during the course of incubation are reported in Fig. 3. The addition of ATP to ADP-supplemented control preparations caused a rapid decrease in oxidation rate and a total cessation of oxygen consumption within 1 hr. (curves B-E). Addition of ATP to ADP- or ATP-supplemented vitamin E-deficient preparations, stopped oxidation only after 2-2x hr. (curves 3-6) and did not lower oxygen consumption to the levels of corresponding controls (curves B-E). The inhibition of oxidation, which depended on the time of ATP addition (curves B-E and 3-6), was not affected by further supplementation with ATP (curves C-D and 4-6), and could not be reversed by subsequent addition of ADP (curve E). Addition of ADP at zero time (curve F), or further ATP supplementation at 1 hr. (curve G), did not affect oxidation by ATP-supplemented control preparations (curve H). DISCUSSION
The increased oxygen consumption by ATP-supplemented washed liver homogenates, during vitamin E deficiency, appeared after the earliest stages of muscular dystrophy, as indicated by increasing urinary creatine excretion and decrease in weight gain by the rabbit. The very large increments in oxygen consumption appeared during stage II (11) of the deficiency. Because of this time factor, the increased oxidation of tricarboxylic acid cycle intermediates, like the increased liver xanthine oxidase activity (7), may be secondary to some more fundamental effect of the deficiency. We have no explanation for the unique behavior of added citrate, as compared to the other substrates investigated, except to note the report of Christie and Judah (19) that the oxidation of added citrate may differ from the oxidation of citrate formed intramitochrondrially. Although substitution of ADP for ATP increased the oxygen consumption of control liver preparations to that of the deficient group, the
EFFECT
OF
VITAMIN
E DEFICIENCY
503
present evidence does not indicate that the difference observed with ATP-supplemented preparations was due to a greater availability of high-energy phosphate-bond acceptor systems during vitamin E deficiency. On the basis of reports concerning the myokinase and adenosinetriphosphatase (ATPase) activity of various liver preparations, “inhibition” of oxygen consumption due to saturation of high-energy phosphate-bond acceptor systems, or decrease of inorganic phosphate in the incubation mixtures, would not be expected with the type of preparations used in these studies (20-28). Morgulis and Jacobi (29) at one time did suggest that the increased respiration of skeletal muscle during vitamin E deficiency might be due to increased ATPase activity. However they (30) and other investigators (31-32) were unable to detect any increase in ATPase activity during the deficiency. Vitamin E deficiency also had no effect on ATPase activity of liver preparations under conditions similar to those employed in the studies reported here (33). In the investigations being reported, the addition of fluoride to a final concentration of 1.5 X 1W2 M, which should have an appreciable effect on ,4TPase and myokinase activity (12, 20, 21, 27, 34, 35), did not markedly reduce the total oxygen consumption by ATP-supplemented deficient liver preparations to control levels. It was also found that oxygen consumption by both control and vitamin E-deficient liver preparations supplemented with AMP was at the same high level as ADP-supplemented systems. Oxygen consumption by controls, then, does not appear to be limited by myokinase activity which is probably unaffected by the deficiency. Finally, supplementation of control preparations with a mixture of ATP and ADP (Fig. 3, F) did not increase oxygen consumption over controls containing only ATP (Fig. 3, H). This was contrary to the result that might be expected if the increase in oxygen consumption obtained by substituting ADP for ATP was due to the ability of ADP to serve as a high-energy phosphate-bond acceptor system. The difference in oxygen consumption by washed liver homogenates during vitamin E deficiency depended on the presence of ATP in the incubation system. Addition of ATP during the incubation resulted in a rapid decrease and complete cessation of oxygen consumption by ADPsupplemented control preparations (Fig. 3, B-E), which could not be reversed by subsequent addition of ADP (Fig. 3, E). Addition of ATP during the incubation of vitamin E-deficient preparations had a much smaller inhibitory effect (Fig. 3, 3-6) and oxygen consumption was not reduced to the levels of corresponding controls (Fig. 3, B-E). The finding
504
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SHOICHET,
GOLDRICH
AND
MILHORAT
that addition of ATP during the incubation of control preparations did not increase oxygen consumption and, in fact, caused its cessation, indicated that the difference in oxidation was not due to a more rapid destruction of ATP. These results suggest that during vitamin E deficiency there is a defect in a mechanism controlling oxidation by washed liver homogenates. Such a control mechanism appears to be dependent on the presence of ATP in the incubation system and independent of the level of highenergy phosphate-bond acceptor systems, at least as represented by ADP.6 A mechanism controlling oxidation, other than by the level of phosphate-bond acceptor systems, has been reported by Johnson and Ackermann (36) and Christie and Judah (19). These investigators have found that the addition of nuclei stimulated oxidation by mitochondria by some means other than the effect of the nuclei on the levels of acceptor systems and to a greater extent than added hexokinase and glucose. Although there is no direct experimental evidence, it is tempting to speculate that vitamin E deficiency may affect yet another mechanism controlling oxidation that is independent of the levels of phosphate-bond acceptor systems. SUMMARY
Oxidation of five of six added tricarboxylic acid substrates by washed liver homogenates from vitamin E-deficient rabbits was 60-600 % higher than controls when supplemented with ATP. Fluoride did not reduce oxidation to control levels. Replacing ATP with ADP or AMP eliminated the difference in oxygen consumption. REFERENCES 1. VICTOR,
J., Am. J. Physiol. 106,229 (1934). MADSEN, L. L., J. Nutrition 11, 471 (1936). FRIEDMAN, I., AND MATILL, H. A., Am. J. Physiol. 131, 595 (1941). HOUCHIN, 0. B., AND MATILL, H. A., J. Biol. Chem. 146, 301 (1942). BASINSKI, D. H., AND HUMMEL, J. P., J. Biol. Chem. 167, 339 (1947). HIJMMEL, J. P., AND BASINSKI, D. H., J. BioZ. Chem. 172, 417 (1948). DINNING, J. S., J. BioZ. Chem. 202, 213 (1953). RICHERT, D. A., AND WESTERFELD, W. W., Proc. Sot. Exptl. Biol. Med. 468 (1953). 9. ROSENKRANTZ, H., J. BioZ. Chem. 214, 789 (1955).
2. 3. 4. 5. 6. 7. 8.
tions
6 In a few experiments the addition of hexokinase and glucose did not increase oxygen consumption (unpublished results).
to these
prepara-
84,
EFFECT
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
OF
VITAMIN
E
DEFICIENCY
505
GOETTSCH, M., ASD PAPPENHEIMER, A. M., J. Erpt2. &fed. 64, 145 (1931). MACKENZIE, C. G., AND MCCOLLUM, E. V., ,J. IY~ttrition 19, 345 (1940). POTTER, V. It., J. Biol. Chenz. 169, 17 (1947). ScHNEIr~ER, W. C., J. Biol. Chem. 176, 259 (1948). HOGEBOO~I, G. H., SCHNEIDER, W. C., AND STREIBICII, hI. .J., J. HioZ. Chem. 196, 111 (1952). SCHSEII~ER, W. C., J. Biol. Chem. 166, 585 (1946). HOGEBOOM, G. H., SCHNEIDER, W. C., AND PALLAUE, G. E., J. Biol. Chews. 172, 619 (1948). HARMOS, J. W., AND OSBORNE, U. H., J. Exptl. h!ied. 98, 81 (1953). HARMOS, J. W., AND FEIGELSON, M., Exptl. Cell Research 3, 509 (1952). CIIRISTIE, G. S., AND JUDAH, J. D., Proc. Roy. Sot. (London) B141,420 (1953). CROSS, R. J., TAGGART, J. V., Covo, G. A., .