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Effect of iron deficiency on energy conservation in rat liver and skeletal muscle submitochondrial particles
BIOCHEMICAL
MEDICINE
34,
93-99
(1985)
Effect of Iron Deficiency on Energy Conservation in Rat Liver and Skeletal Muscle Submitochondrial Particles TIMOTHY
C. EVANS AND BRUCE MACKLER
Departments of Medicine and Pediatrics and the Center for Child Development and Mental Retardation, University of Washington School qf Medicine, Seattle, Washington 98195 Received
May
7, 1984
It is well documented that iron deficiency not only causes anemia but results in diminished levels of iron-containing tissue enzymes as well (l-7). Evaluation of mitochondrial function has shown that the rate of electron flow through NADH dehydrogenase, succinate dehydrogenase, and the cytochrome chain is decreased. Those studies which have reported efficiency of coupling of energy conservation to electron transport have shown normal ADP/O ratios in iron-deficient skeletal muscle mitochondria (2,7), heart and liver mitochondria (8), and brain mitochondria (9). This is despite work which has shown marked mitochondrial structural abnormalities in iron deficiency (S&10). Because of the importance of structure in the energy-conserving functions of mitochondria, we undertook the current study to survey more completely energy-linked reactions in preparations from normal and iron-deficient rat liver and skeletal muscle mitochondria. METHODS Animals were made iron deficient as described previously (2,11,12). After 46 weeks, rats were anemic and only those with hemoglobins of 6 g/d1 or less were used. Control animals received the same low-iron diet plus weekly intraperitoneal injections of 5 mg iron dextran. Liver mitochondria were prepared in isotonic sucrose in the standard fashion (13). Liver submitochondrial particles were prepared by sonication after pretreatment with a low concentration digitonin (0. II mg digitonin/mg protein for 20 min) followed by low-power sonication in 0.25 M sucrose, 2 mM EDTA, pH 8.0, in a modification of the procedures of Hoppel and Cooper (14) and Levy et al. (15) as described by T. C. Evans and F. L. Hoch (in preparation). The digitonin step was included to decrease the outer mitochondrial membrane adenylate kinase content of the final submitochondrial particle preparation since adenylate kinase interferes with determination of ATP synthesis by the spectrophotometric method employed here. Skeletal muscle mitochondria were prepared from 15 g wet wt of mixed hind leg muscle as previously described (16,17) but without the addition of ATP to 93 0006-2944/85
$3.00
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction m any form reserved
94
EVANS
AND MACKLEK
the preparative solutions. Mitochondria prepared in this way arc stable for at least several hours and with pyruvate/malate as substrate have a respiratory control index of 6 and ADP/O ratio of 2.8. For submitochondrial particle preparation, the final mitochondrial wash was in 50 mM KH,PO,, pH 7.5, and suspension of the mitochondria1 pellet was in 15 ml of this solution. The mit~chondri~~ were sonicated four times for 1.5 set each using a medium probe of a B. Braun Model 1510 Braunsonic at 300 W with stirring on ice. The sonicate was centrifuged 15 min at 20,000 g and the supernatant recentrifuged 30 min at 100,OOOg. The final pellet of submitochondrial particles was resuspended by gentle homogenization in a solution of 0.25 M sucrose, 1 mM MgCI,, 2 mM EDTA, and 10 mM Tris, pH 7.5. All enzymatic assays were carried out at 30°C in a basic reaction mixture of 0.25 M sucrose. 50 mM Tris acetate, 4 mM MgSOJ, pH 8.0. except energyindependent transhydrogenation which was in 0.25 M sucrose, 50 m&r KH?PO,, pH 6.5, by the method of Stein et a/. (18). Respiration was measured polarographically in the presence of IO mM KH,PO., with a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs. Ohio). Energy-dependent transhydrogenation was measured by the method of Rydstrom et (II. (19) and reversed electron flow (REF)’ (ATP-driven succinate reduction of NAD’), as described by Ernster and Lee (20). ATP synthesis with succinate as substrate was measured spectrophotometricaily (T. C. Evans and F. L. Hoch, in preparation) as the rate of NADPH formation in the basic medium plus 10 mlw KI-IZPO1, 10 mM glucose, 200 PM NADP’, and 12.5 units hexokinaseiglucose-6-phosphate dehydrogenase (Sigma H-8629, diluted 200 units/O.32 ml 0.1 M Tris. 4 mMEDTA. 1% glucose. pH 7.4). Since in this assay procedure ATP generation by myokinase (2 ADP + AMP + ATP) is also detected, sufficient time was allowed after the addition of I mM ADP to establish this rate (control liver submitochondrial particles. 17.2 i: 1.7 nmof ATP/min/mg; iron deficient, t8.3 2: 2.3 nmoi ATP/min/mg) and it was subtracted from the total rate of ATP formation after subsequent succinate addition to determine succinate-supported ATP synthesis. ATPase was measured by the method of Stiggall et (I/. (21). Substrate concentrations in the various assays were succinate, 10 mM, NADH, 0.X tn~, P-hydroxybutyrate. 20 mnf. plus NAD*, 2 mM, and cr-glycerophosphate, 26 mM. Cytochrome difference spectra (dithionite reduced minus oxidized) were recorded with an American Instrument DW-2 uv-vis spectrophotometer and cytochrome contents calcuated from the foliowing wavelength pairs and mil~imofar extinction coefficients: cytochrome 6 (562-575 nm), 20.0 (22): cytochromes r’ + f’, (550540 nm), 19.1 (23): cytochromes u + + (605-630 nm). 24.0 (24). Protein concentrations were determined by the procedures of Got-nail (‘I (ri. (2.5) and Lowry et nl. (26). ’ Abbreviations used: REF. reversed electron Row (A’We-energized succinate reduction of N&If I: TH, energy-independent t~ns~ydrogenation (NADPH -+ NAD’ -+ NADP’ -1.UADH,: ETH,,~,\ ix ETH,w energy-dependent trans~ydrogenation driven by succinatr ~~i~~li~~n or .%TP ~yLir~~~~i~is (NADH + NADP’ + - --+ NAD’ * NADPH): ADP -+ ATP. succinate-driven ph~~~p~ory~ation of ADP to ATP: succ. - succinate: &OHB, ,&hydroxybutyrate: wGP, (y-RIycel;OphOsphate; oligo. oligomycin.
MITOCHONDRIA
IN IRON DEFICIENCY
9.5
Statistical analysis was by standard methods (27). All results are presented as the mean t standard error of the mean. Calculation of the ratio + standard error of two means + standard error and determination of the significance of the difference between two such calculated ratios were by the methods of Finney (28) and Cochran (29), respectively. RESULTS Liver. In the studies performed, there is little difference in the results obtained between control and iron-deficient liver submitochondrial particles (Tables 1 and 2). Despite marked anemia, there was no apparent effect on cytochrome content or respiration rate during oxidation of P-hydroxybutyrate/NAD’ or succinate. Both these oxidase systems involve nonheme iron centers (mitochondrial complexes I and II, respectively) as well as the cytochromes. A significant though slight (16%) decrease was seen in ATP-driven energy-dependent transhydrogenation. The rates of those energy-linked processes involving succinate oxidation (REF, ETH succ, and ADP -+ ATP) were normal and the calculated ratios of these TABLE Liver Submitochondrial
.__-____ Respiration (nmol O/min/mg) _--____ succ /%OHB/NAD+
Hemoglobin (g/dU -.~--~. Control tN = 7) 14.6 c 0.4
188.9 + 14.9
Fe deficient (N = 7) 5.3 166.2 2 0.3 t 3.5 P < 0.001
NS
1 Particle Function
REF
TH
175.9 + 14.2
71.2 r 5.5
52.6 ‘- 5.7
171.5 5.9
82.2 i 7.0
43.8 2 3.1
NS
NS
t
-~
NS
ET&,,
ETHm
nmol/min/mg 68.4 113.3 -+ 3.5 2 6.6 62.5 + 1.7 NS .~
ADP-+ATP
103.6 t- 6.4
95.0 t 4.1
98.1 i 4.3
P < 0.05
NS
TABLE 2 Particle Cytochrome Content
Submitochondrial
Cytochrome (nmol/mg)
Liver Control (N = 6) Fe deficient (N = 7) Muscle Control (N = 5) Fe deficient (N = 4)
b
c + Cl
0.31 k 0.01 0.24 2 0.02 NS
0.48 ” 0.00 0.49 * 0.03 NS
0.24 2 0.01 0.25 2 0.01 NS
0.63 2 0.03 0.24 k 0.02 P < 0.001
0.51 k 0.02 0.24 f 0.03 P < 0.001
0.57 2 0.02 0.48 2 0.06 NS
a +
a,
96
EVANSANDMACKLER
succinate-driven reaction rates to the rate of succinate respiration (analogous to the mitochondrial P/O ratio) and determination of the significance of the differences between normal and iron-deficient in these calculated ratios revealed a significant change from control to iron-deficient groups only of the REF:respiration ratio (0.379 _t 0.018 to 0.494 i 0.041. P < 0.05). There was no control to irondeficient change in either the ETH,,,,:respiration ratio (0.369 i 0.024 to 0.383 t 0.012) or the ADP * ATP:respiration ratio (0.563 f 0.045 to 0.593 i 0.033). Skeletal muscle. In contrast to liver, submitochondrial particles from irondeficient skeletal muscle mitochondria were distinctly abnormal in cytochrome content and enzymatic function (Tables 2 and 3). Cytochromes h and (’ i (‘r were significantly decreased, by 62 and 53%, respectively. The respiration rates with NADH and succinate were decreased by 86 and 82%. respectively. Energylinked reactions depending on succinate oxidation (REF and ETH,,,,,) were also dramatically reduced. The transhydrogenase enzyme itself and the ATPase complex and coupling mechanism seemed to be normal as suggested by unchanged rates of energy-independent transhydrogenation. ATP-driven energy-dependent transhydrogenation, and oligomycin-sensitive AT&se. (Succinate-driven ATP synthesis could not be observed in this submitochondrial particle preparation for either control or iron-deficient skeletal muscle.) DISCUSSION To date, most studies of the effects of iron deficiency on mammalian mitochondria have documented decreased amounts of iron-containing mitochondrial components as a consequence of iron deficiency. The easiest components to measure and quantitate are the cytochromes and many reports have shown their negative response to iron deficiency in a variety of tissues (I-7). More recently, the iron of rat skeletal muscle mitochondrial iron-sulfur centers has been evaluated (7) and specific centers in both complexes I and II, as well as electron-transfer flavoprotein are decreased in iron deficiency. A number of workers have also shown that the electron-transferring capacity of these various components is correspondingly decreased in iron deficiency. Previous studies (2,7-9) which have evaluated the energy-conserving capability of mitochondria in iron deficiency have shown that though the rate of ATP synthesis is decreased, the efficiency (ADPIO ratio) of phosphorylation is unchanged. Thus. the abnormality in mitochondrial function produced by iron deficiency would appear to occur at the level of the substate oxidase systems, a view with which our current data are entirely consistent. Of the two tissues examined here, skeletal muscle is decidedly more affected by iron deficiency than liver. Liver submitochondrial particles prepared from iron-deficient rats show neither an abnormality in cytochrome content nor more than a minor change in any of several dehydrogenases/oxidases, transhydrogenase or energy-conserving reactions. Yet in skeletal muscle mitochondria. all cytochromes are affected (b > c + c, > (I +- (I?) and respiration through complexes I and II was decreased 85%. Energy-conserving processes which required participation of a dehydrogenase system (REF and ETH,,,,) were similarly decreased
MITOCHONDRIA
IN
IRON
DEFICIENCY
97
98
EVANS AND MACKLER
while ATP-driven transhydrogenation and mitochondrial ATPase activity were unchanged in iron deficiency. Our survey of a variety of energy-Iinked reactions suggests that though ahnormalities of mitochondrial size and shape have been reported in iron deficiency (5,8,10), raising the possibility of abnormal coupling of electron transport to energy conservation, the abnormal mito~hondrial function in iron deficiency seems limited to a decreased carrying capacity of iron-containing electron-tranhferring components while the ability of mitochondria to utitize the generated free energy potential is normal. Our data emphasize. however, that in tissues where electron transport is decreased by iron deficiency (e.g.. skeletal muscle), energydependent reactions which depend on those oxidations (energy-linked transhydrogenation and reversed electron flow) are markedly decreased. SUMMARY
S~bmitochondria~ particles prepared from liver and skeletal muscle of control and iron-deficient rats were examined for cytochrome content and for both energyindependent and energy-conserving functions. Liver submitochondrial particles appear quite resistant to iron deficiency with cytochrome content and electrontransferring or energy-conserving functions maintained at a level of XW or better of normal. Iron-deficient skeletal muscle submitochondrial particles, in contrast, have decreased ~yto~hrome content and only 12-20% of the normal capacity for oxidation through either complex I (NADH dehydrogenase) or complex 11 (succinate dehydrogenase). Energy-linked reactions which involve substrate oxidationireduction (succinate + NAD” reversed electron flow and succinate-driven energydependent transhydrogenation) are likewise markedly decreased. while ATPdriven energy-dependent transhydrogenation and mitochondrial ATPdse are normai. Our data support the concept that iron deficiency leads to decreased electroncarrying capacity of iron-containing mitochondria~ enzymes. with skeletal muscle being much more susceptible than liver, but that the mitochondria are otherwise normal with regard to energy conservation. ACKNOWLEDGMENTS and
This work was supported in part by National Institutes of Health Grants HD 02274. T.C.E. was the recipient of NIH postdoctoral traineeship.
HD 15Yl.Ss AM 07237.
GM
1?300h.
REFERENCES 1. Dallman, P. R., Beutler, E., and Finch, C. A.. &r/r. ‘1. ff~f~t??uf(~i. 4% 179 (1978). 2. Finch. C. A., Miller. L. R.. Inamdar, A. R.. Person, R. Seiler. K.. and Mackler, Invesf.
58,
R., .i. C&j.
447 (1976).
3. Finch, C. A.. Gollnick, P. D., fllastala. M. P.. Miller. L. K.. Dillman, E.. and Mackler-. B.. 3. Clin.
Invest.
64, 129 (1979).
4. Dallman, P. R., and Schwartz, H. C.. P~~i~~r~~~~~ 35. 677 (l9h.S). 5. Dallman, P. R., and Goodman, J. R., J. Cell B&J!. 48, 79 I 1971). 6. McLane, J. A., Fell. R. D., McKay, R. H.. Winder. W. W.. Brown. rlmer. 7.
J. Physiol.
Maguire, 210
J. J..
E. B.. and
Hotlosq.
Davies, K. J. A., Daiiman. P. IL, and Packer. L..
Bicrchim.
Rioplt~.~
(1982).
8. Goodman,
J.
J. il.
241, C47 11981).
R., Warshaw. J. B.. and Dallman. I’. K.. Pedrc~~. fte~s.4. 244 (19701.
km
679,
MITOCHONDRIA
IN IRON
DEFICIENCY
99
9. Mackler. B.. Person, R.. Miller, L. R.. Inamdar, A. R.. and Finch. C. A.. Pediatr. Res.12, 1-17 (1978).
10. Dallman. P. R.. and Goodman, J. R., Blood 35, 496-505 (1970). 11. Dillman, E., Johnson, D. G.. Martin, J.. Mackler. B.. and Finch. C. A.. Amer. J. Physiol. 237. R297
(1979).
12. Dillman, E., Gale, C., Green, W.. Johnson, D. G., Mackler. B.. and Finch. C.. Amer. J. Physiol. 239, R377
(1980).
Hoch, F. L.. Endocrinology 77, 991 (1965). 14. Hoppel, C., and Cooper, C.. Biochem. J. 107, 367 (1968). IS;. Levy, M.. Toury, R., and Andre. J., Biochim. Biophys. Actu 135. 599 (1967). 16. Makinen. M. W.. and Lee, C.-P., Arch. Biochem. Biophys. 126, 75 (1968). 17. Lee. C.-P., Martens, M. E., Jankulovska. L.. and Neymark, M. A., Muscle Nenje 2, 340 (1979). 18. Stein, A. M.. Kaplan, N. 0.. and Ciotti. M. M., J. Biol. Chem. 234, 979 (1959). 19. Rydstrom. J., Teixeira Da Cruz, A., and Ernster. L., Eur. J. Biochem. 23, 212 (1971). 20. Ernster, L., and Lee, C.-P., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman. Eds.), Vol. 10. p. 729. Academic Press, New York, 1967. 21. Stiggall, D. L., Galante. Y. M.. and Hatefi. Y., in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 55, p. 308. Academic Press, New York, 1979. 22. Chance, B.. and Williams. G. R., J. Biol. Chem. 217, 395 (1955). 23. Chance, B.. Nature (London) 169, 215 (1952). 24. Van Gelder. B. F., Biochim. Biophys. Acta 118, 36 (1966). 25. Gornall, A. G., Bardawill, C. J.. and David, M. M.. J. Biol. Chetn. 177, 751 (1949). 26. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L.. and Randall, R. J.. J. Biol. Chem. 193, 265 13.
(1951).
Snedecor. G. W.. and Cochran, W. G.. “Statistical Methods.” 6th ed., p. 258. Iowa State Univ. Press, Ames, 1974. 28. Finney. D. J.. “Statistical Methods in Biological Assays,” p. 24-29. Hafner, New York, 1964. 29. Cochran, W. G.. “Sampling Techniques,” 2nd ed., p. 181. Wiley. New York. 1963. 27.
MEDICINE
34,
93-99
(1985)
Effect of Iron Deficiency on Energy Conservation in Rat Liver and Skeletal Muscle Submitochondrial Particles TIMOTHY
C. EVANS AND BRUCE MACKLER
Departments of Medicine and Pediatrics and the Center for Child Development and Mental Retardation, University of Washington School qf Medicine, Seattle, Washington 98195 Received
May
7, 1984
It is well documented that iron deficiency not only causes anemia but results in diminished levels of iron-containing tissue enzymes as well (l-7). Evaluation of mitochondrial function has shown that the rate of electron flow through NADH dehydrogenase, succinate dehydrogenase, and the cytochrome chain is decreased. Those studies which have reported efficiency of coupling of energy conservation to electron transport have shown normal ADP/O ratios in iron-deficient skeletal muscle mitochondria (2,7), heart and liver mitochondria (8), and brain mitochondria (9). This is despite work which has shown marked mitochondrial structural abnormalities in iron deficiency (S&10). Because of the importance of structure in the energy-conserving functions of mitochondria, we undertook the current study to survey more completely energy-linked reactions in preparations from normal and iron-deficient rat liver and skeletal muscle mitochondria. METHODS Animals were made iron deficient as described previously (2,11,12). After 46 weeks, rats were anemic and only those with hemoglobins of 6 g/d1 or less were used. Control animals received the same low-iron diet plus weekly intraperitoneal injections of 5 mg iron dextran. Liver mitochondria were prepared in isotonic sucrose in the standard fashion (13). Liver submitochondrial particles were prepared by sonication after pretreatment with a low concentration digitonin (0. II mg digitonin/mg protein for 20 min) followed by low-power sonication in 0.25 M sucrose, 2 mM EDTA, pH 8.0, in a modification of the procedures of Hoppel and Cooper (14) and Levy et al. (15) as described by T. C. Evans and F. L. Hoch (in preparation). The digitonin step was included to decrease the outer mitochondrial membrane adenylate kinase content of the final submitochondrial particle preparation since adenylate kinase interferes with determination of ATP synthesis by the spectrophotometric method employed here. Skeletal muscle mitochondria were prepared from 15 g wet wt of mixed hind leg muscle as previously described (16,17) but without the addition of ATP to 93 0006-2944/85
$3.00
Copyright 0 1985 by Academic Press. Inc. All rights of reproduction m any form reserved
94
EVANS
AND MACKLEK
the preparative solutions. Mitochondria prepared in this way arc stable for at least several hours and with pyruvate/malate as substrate have a respiratory control index of 6 and ADP/O ratio of 2.8. For submitochondrial particle preparation, the final mitochondrial wash was in 50 mM KH,PO,, pH 7.5, and suspension of the mitochondria1 pellet was in 15 ml of this solution. The mit~chondri~~ were sonicated four times for 1.5 set each using a medium probe of a B. Braun Model 1510 Braunsonic at 300 W with stirring on ice. The sonicate was centrifuged 15 min at 20,000 g and the supernatant recentrifuged 30 min at 100,OOOg. The final pellet of submitochondrial particles was resuspended by gentle homogenization in a solution of 0.25 M sucrose, 1 mM MgCI,, 2 mM EDTA, and 10 mM Tris, pH 7.5. All enzymatic assays were carried out at 30°C in a basic reaction mixture of 0.25 M sucrose. 50 mM Tris acetate, 4 mM MgSOJ, pH 8.0. except energyindependent transhydrogenation which was in 0.25 M sucrose, 50 m&r KH?PO,, pH 6.5, by the method of Stein et a/. (18). Respiration was measured polarographically in the presence of IO mM KH,PO., with a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs. Ohio). Energy-dependent transhydrogenation was measured by the method of Rydstrom et (II. (19) and reversed electron flow (REF)’ (ATP-driven succinate reduction of NAD’), as described by Ernster and Lee (20). ATP synthesis with succinate as substrate was measured spectrophotometricaily (T. C. Evans and F. L. Hoch, in preparation) as the rate of NADPH formation in the basic medium plus 10 mlw KI-IZPO1, 10 mM glucose, 200 PM NADP’, and 12.5 units hexokinaseiglucose-6-phosphate dehydrogenase (Sigma H-8629, diluted 200 units/O.32 ml 0.1 M Tris. 4 mMEDTA. 1% glucose. pH 7.4). Since in this assay procedure ATP generation by myokinase (2 ADP + AMP + ATP) is also detected, sufficient time was allowed after the addition of I mM ADP to establish this rate (control liver submitochondrial particles. 17.2 i: 1.7 nmof ATP/min/mg; iron deficient, t8.3 2: 2.3 nmoi ATP/min/mg) and it was subtracted from the total rate of ATP formation after subsequent succinate addition to determine succinate-supported ATP synthesis. ATPase was measured by the method of Stiggall et (I/. (21). Substrate concentrations in the various assays were succinate, 10 mM, NADH, 0.X tn~, P-hydroxybutyrate. 20 mnf. plus NAD*, 2 mM, and cr-glycerophosphate, 26 mM. Cytochrome difference spectra (dithionite reduced minus oxidized) were recorded with an American Instrument DW-2 uv-vis spectrophotometer and cytochrome contents calcuated from the foliowing wavelength pairs and mil~imofar extinction coefficients: cytochrome 6 (562-575 nm), 20.0 (22): cytochromes r’ + f’, (550540 nm), 19.1 (23): cytochromes u + + (605-630 nm). 24.0 (24). Protein concentrations were determined by the procedures of Got-nail (‘I (ri. (2.5) and Lowry et nl. (26). ’ Abbreviations used: REF. reversed electron Row (A’We-energized succinate reduction of N&If I: TH, energy-independent t~ns~ydrogenation (NADPH -+ NAD’ -+ NADP’ -1.UADH,: ETH,,~,\ ix ETH,w energy-dependent trans~ydrogenation driven by succinatr ~~i~~li~~n or .%TP ~yLir~~~~i~is (NADH + NADP’ + - --+ NAD’ * NADPH): ADP -+ ATP. succinate-driven ph~~~p~ory~ation of ADP to ATP: succ. - succinate: &OHB, ,&hydroxybutyrate: wGP, (y-RIycel;OphOsphate; oligo. oligomycin.
MITOCHONDRIA
IN IRON DEFICIENCY
9.5
Statistical analysis was by standard methods (27). All results are presented as the mean t standard error of the mean. Calculation of the ratio + standard error of two means + standard error and determination of the significance of the difference between two such calculated ratios were by the methods of Finney (28) and Cochran (29), respectively. RESULTS Liver. In the studies performed, there is little difference in the results obtained between control and iron-deficient liver submitochondrial particles (Tables 1 and 2). Despite marked anemia, there was no apparent effect on cytochrome content or respiration rate during oxidation of P-hydroxybutyrate/NAD’ or succinate. Both these oxidase systems involve nonheme iron centers (mitochondrial complexes I and II, respectively) as well as the cytochromes. A significant though slight (16%) decrease was seen in ATP-driven energy-dependent transhydrogenation. The rates of those energy-linked processes involving succinate oxidation (REF, ETH succ, and ADP -+ ATP) were normal and the calculated ratios of these TABLE Liver Submitochondrial
.__-____ Respiration (nmol O/min/mg) _--____ succ /%OHB/NAD+
Hemoglobin (g/dU -.~--~. Control tN = 7) 14.6 c 0.4
188.9 + 14.9
Fe deficient (N = 7) 5.3 166.2 2 0.3 t 3.5 P < 0.001
NS
1 Particle Function
REF
TH
175.9 + 14.2
71.2 r 5.5
52.6 ‘- 5.7
171.5 5.9
82.2 i 7.0
43.8 2 3.1
NS
NS
t
-~
NS
ET&,,
ETHm
nmol/min/mg 68.4 113.3 -+ 3.5 2 6.6 62.5 + 1.7 NS .~
ADP-+ATP
103.6 t- 6.4
95.0 t 4.1
98.1 i 4.3
P < 0.05
NS
TABLE 2 Particle Cytochrome Content
Submitochondrial
Cytochrome (nmol/mg)
Liver Control (N = 6) Fe deficient (N = 7) Muscle Control (N = 5) Fe deficient (N = 4)
b
c + Cl
0.31 k 0.01 0.24 2 0.02 NS
0.48 ” 0.00 0.49 * 0.03 NS
0.24 2 0.01 0.25 2 0.01 NS
0.63 2 0.03 0.24 k 0.02 P < 0.001
0.51 k 0.02 0.24 f 0.03 P < 0.001
0.57 2 0.02 0.48 2 0.06 NS
a +
a,
96
EVANSANDMACKLER
succinate-driven reaction rates to the rate of succinate respiration (analogous to the mitochondrial P/O ratio) and determination of the significance of the differences between normal and iron-deficient in these calculated ratios revealed a significant change from control to iron-deficient groups only of the REF:respiration ratio (0.379 _t 0.018 to 0.494 i 0.041. P < 0.05). There was no control to irondeficient change in either the ETH,,,,:respiration ratio (0.369 i 0.024 to 0.383 t 0.012) or the ADP * ATP:respiration ratio (0.563 f 0.045 to 0.593 i 0.033). Skeletal muscle. In contrast to liver, submitochondrial particles from irondeficient skeletal muscle mitochondria were distinctly abnormal in cytochrome content and enzymatic function (Tables 2 and 3). Cytochromes h and (’ i (‘r were significantly decreased, by 62 and 53%, respectively. The respiration rates with NADH and succinate were decreased by 86 and 82%. respectively. Energylinked reactions depending on succinate oxidation (REF and ETH,,,,,) were also dramatically reduced. The transhydrogenase enzyme itself and the ATPase complex and coupling mechanism seemed to be normal as suggested by unchanged rates of energy-independent transhydrogenation. ATP-driven energy-dependent transhydrogenation, and oligomycin-sensitive AT&se. (Succinate-driven ATP synthesis could not be observed in this submitochondrial particle preparation for either control or iron-deficient skeletal muscle.) DISCUSSION To date, most studies of the effects of iron deficiency on mammalian mitochondria have documented decreased amounts of iron-containing mitochondrial components as a consequence of iron deficiency. The easiest components to measure and quantitate are the cytochromes and many reports have shown their negative response to iron deficiency in a variety of tissues (I-7). More recently, the iron of rat skeletal muscle mitochondrial iron-sulfur centers has been evaluated (7) and specific centers in both complexes I and II, as well as electron-transfer flavoprotein are decreased in iron deficiency. A number of workers have also shown that the electron-transferring capacity of these various components is correspondingly decreased in iron deficiency. Previous studies (2,7-9) which have evaluated the energy-conserving capability of mitochondria in iron deficiency have shown that though the rate of ATP synthesis is decreased, the efficiency (ADPIO ratio) of phosphorylation is unchanged. Thus. the abnormality in mitochondrial function produced by iron deficiency would appear to occur at the level of the substate oxidase systems, a view with which our current data are entirely consistent. Of the two tissues examined here, skeletal muscle is decidedly more affected by iron deficiency than liver. Liver submitochondrial particles prepared from iron-deficient rats show neither an abnormality in cytochrome content nor more than a minor change in any of several dehydrogenases/oxidases, transhydrogenase or energy-conserving reactions. Yet in skeletal muscle mitochondria. all cytochromes are affected (b > c + c, > (I +- (I?) and respiration through complexes I and II was decreased 85%. Energy-conserving processes which required participation of a dehydrogenase system (REF and ETH,,,,) were similarly decreased
MITOCHONDRIA
IN
IRON
DEFICIENCY
97
98
EVANS AND MACKLER
while ATP-driven transhydrogenation and mitochondrial ATPase activity were unchanged in iron deficiency. Our survey of a variety of energy-Iinked reactions suggests that though ahnormalities of mitochondrial size and shape have been reported in iron deficiency (5,8,10), raising the possibility of abnormal coupling of electron transport to energy conservation, the abnormal mito~hondrial function in iron deficiency seems limited to a decreased carrying capacity of iron-containing electron-tranhferring components while the ability of mitochondria to utitize the generated free energy potential is normal. Our data emphasize. however, that in tissues where electron transport is decreased by iron deficiency (e.g.. skeletal muscle), energydependent reactions which depend on those oxidations (energy-linked transhydrogenation and reversed electron flow) are markedly decreased. SUMMARY
S~bmitochondria~ particles prepared from liver and skeletal muscle of control and iron-deficient rats were examined for cytochrome content and for both energyindependent and energy-conserving functions. Liver submitochondrial particles appear quite resistant to iron deficiency with cytochrome content and electrontransferring or energy-conserving functions maintained at a level of XW or better of normal. Iron-deficient skeletal muscle submitochondrial particles, in contrast, have decreased ~yto~hrome content and only 12-20% of the normal capacity for oxidation through either complex I (NADH dehydrogenase) or complex 11 (succinate dehydrogenase). Energy-linked reactions which involve substrate oxidationireduction (succinate + NAD” reversed electron flow and succinate-driven energydependent transhydrogenation) are likewise markedly decreased. while ATPdriven energy-dependent transhydrogenation and mitochondrial ATPdse are normai. Our data support the concept that iron deficiency leads to decreased electroncarrying capacity of iron-containing mitochondria~ enzymes. with skeletal muscle being much more susceptible than liver, but that the mitochondria are otherwise normal with regard to energy conservation. ACKNOWLEDGMENTS and
This work was supported in part by National Institutes of Health Grants HD 02274. T.C.E. was the recipient of NIH postdoctoral traineeship.
HD 15Yl.Ss AM 07237.
GM
1?300h.
REFERENCES 1. Dallman, P. R., Beutler, E., and Finch, C. A.. &r/r. ‘1. ff~f~t??uf(~i. 4% 179 (1978). 2. Finch. C. A., Miller. L. R.. Inamdar, A. R.. Person, R. Seiler. K.. and Mackler, Invesf.
58,
R., .i. C&j.
447 (1976).
3. Finch, C. A.. Gollnick, P. D., fllastala. M. P.. Miller. L. K.. Dillman, E.. and Mackler-. B.. 3. Clin.
Invest.
64, 129 (1979).
4. Dallman, P. R., and Schwartz, H. C.. P~~i~~r~~~~~ 35. 677 (l9h.S). 5. Dallman, P. R., and Goodman, J. R., J. Cell B&J!. 48, 79 I 1971). 6. McLane, J. A., Fell. R. D., McKay, R. H.. Winder. W. W.. Brown. rlmer. 7.
J. Physiol.
Maguire, 210
J. J..
E. B.. and
Hotlosq.
Davies, K. J. A., Daiiman. P. IL, and Packer. L..
Bicrchim.
Rioplt~.~
(1982).
8. Goodman,
J.
J. il.
241, C47 11981).
R., Warshaw. J. B.. and Dallman. I’. K.. Pedrc~~. fte~s.4. 244 (19701.
km
679,
MITOCHONDRIA
IN IRON
DEFICIENCY
99
9. Mackler. B.. Person, R.. Miller, L. R.. Inamdar, A. R.. and Finch. C. A.. Pediatr. Res.12, 1-17 (1978).
10. Dallman. P. R.. and Goodman, J. R., Blood 35, 496-505 (1970). 11. Dillman, E., Johnson, D. G.. Martin, J.. Mackler. B.. and Finch. C. A.. Amer. J. Physiol. 237. R297
(1979).
12. Dillman, E., Gale, C., Green, W.. Johnson, D. G., Mackler. B.. and Finch. C.. Amer. J. Physiol. 239, R377
(1980).
Hoch, F. L.. Endocrinology 77, 991 (1965). 14. Hoppel, C., and Cooper, C.. Biochem. J. 107, 367 (1968). IS;. Levy, M.. Toury, R., and Andre. J., Biochim. Biophys. Actu 135. 599 (1967). 16. Makinen. M. W.. and Lee, C.-P., Arch. Biochem. Biophys. 126, 75 (1968). 17. Lee. C.-P., Martens, M. E., Jankulovska. L.. and Neymark, M. A., Muscle Nenje 2, 340 (1979). 18. Stein, A. M.. Kaplan, N. 0.. and Ciotti. M. M., J. Biol. Chem. 234, 979 (1959). 19. Rydstrom. J., Teixeira Da Cruz, A., and Ernster. L., Eur. J. Biochem. 23, 212 (1971). 20. Ernster, L., and Lee, C.-P., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman. Eds.), Vol. 10. p. 729. Academic Press, New York, 1967. 21. Stiggall, D. L., Galante. Y. M.. and Hatefi. Y., in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. 55, p. 308. Academic Press, New York, 1979. 22. Chance, B.. and Williams. G. R., J. Biol. Chem. 217, 395 (1955). 23. Chance, B.. Nature (London) 169, 215 (1952). 24. Van Gelder. B. F., Biochim. Biophys. Acta 118, 36 (1966). 25. Gornall, A. G., Bardawill, C. J.. and David, M. M.. J. Biol. Chetn. 177, 751 (1949). 26. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L.. and Randall, R. J.. J. Biol. Chem. 193, 265 13.
(1951).
Snedecor. G. W.. and Cochran, W. G.. “Statistical Methods.” 6th ed., p. 258. Iowa State Univ. Press, Ames, 1974. 28. Finney. D. J.. “Statistical Methods in Biological Assays,” p. 24-29. Hafner, New York, 1964. 29. Cochran, W. G.. “Sampling Techniques,” 2nd ed., p. 181. Wiley. New York. 1963. 27.