Perturbation of mitochondrial composition in muscle by iron deficiency. Implications regarding regulation of mitochondrial assembly

It has been reported that the mitochondrial cytochromes and citrate cycle enzymes occur in constant proportions to each other and increase or decrease roughly in parallel in response to various stimuli. The purpose of this study was to determine whether this proportionality is an obligatory consequence of the way in which mitochondria are assembled. Severe iron deficiency was used to bring about decreases of the iron-containing constituents of the mitochondrial respiratory chain in skeletal muscle. Cytochrome c concentration and cytochrome oxidase activity were decreased approximately 50%, while succinate dehydrogenase and NADH dehydrogenase activities were decreased by 78% in iron-deficient muscle. On electron microscopic examination, mitochondria in iron-deficient muscles had relatively sparse numbers of cristae. The iron deficiency had little or no effect on the levels of a range of mitochondrial matrix enzymes, including citrate synthase, isocitrate dehydrogenase, fumarase, aspartate aminotransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and acetoacetyl-CoA thiolase. These results show that the usual constant proportions between the constituents of the mitochondrial respiratory chain and matrix enzymes are not obligatory; they provide evidence that mitochondrial matrix enzymes and respiratory chain constituents can be incorporated into mitochondria independently and that the ratios between them can vary within wide limits.     

Physiological and biochemical correlates of increased work in trained iron-deficient rats

We investigated physiological and biochemical factors associated with the improved work capacity of trained iron-deficient rats. Female 21-day-old rats were assigned to one of four groups, two dietary groups (50 and 6 ppm dietary iron) subdivided into two levels of activity (sedentary and treadmill trained). Iron deficiency decreased hemoglobin (61%), maximal O2 uptake. (VO2max) (40%), skeletal muscle mitochondrial oxidase activities (59-90%), and running endurance (94%). In contrast, activities of tricarboxylic acid (TCA) cycle enzymes in skeletal muscle were largely unaffected. Four weeks of mild training in iron-deficient rats resulted in improved blood lactate homeostasis during exercise and increased VO2max (15%), TCA cycle enzymes of skeletal muscle (27-58%) and heart (29%), and liver NADH oxidase (34%) but did not affect any of these parameters in the iron-sufficient animals. In iron-deficient rats training affected neither the blood hemoglobin level nor any measured iron-dependent enzyme pathway of skeletal muscle but substantially increased endurance (230%). We conclude that the training-induced increase in endurance in iron-deficient rats may be related to cardiovascular improvements, elevations in liver oxidative capacity, and increases in the activities of oxidative enzymes that do not contain iron in skeletal and cardiac muscle.     

Tissue effects of iron deficiency in the rat

Measurements of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase activities, iron, cytochrome c and myoglobin, were made on various hind-leg muscles, fast-twitch red and white muscle and heart and liver of male Wistar rats fed an iron-deficient diet on weaning. Rats fed the same diet and given 20 mg iron intraperitoneally as iron-dextran (Imferon) served as controls. For iron-repletion studies anemic rats (hemoglobin less than 7 g/dl) were given a single injection of 10 mg iron (Imferon) and the time course of change in the above parameters was followed up to 22 days after injection. The iron concentration of most iron-deficient muscles dropped to approx. 35% of control, the heart to 60% and liver to 13%. On repletion, the iron concentration of all tissues increase significantly by 4 days. While the levels of cytochrome c and myoglobin approximated the iron levels in muscle, they did not change significantly in the heart. Succinate dehydrogenase activity dropped profoundly in muscle, to 10-30% of control; on repletion, the activity increased significantly. Mitochondrial glycerol-3-phosphate dehydrogenase activity showed only small changes in iron-deficient tissues.     

Mild Hyperbaric Oxygen Improves Decreased Oxidative Capacity of Spinal Motoneurons Innervating the Soleus Muscle of Rats with Type 2 Diabetes

Rats with type 2 diabetes exhibit decreased oxidative capacity, such as reduced oxidative enzyme activity, low-intensity staining for oxidative enzymes in fibers, and no high-oxidative type IIA fibers, in the skeletal muscle, especially in the soleus muscle. In contrast, there are no data available concerning the oxidative capacity of spinal motoneurons innervating skeletal muscle of rats with type 2 diabetes. This study examined the oxidative capacity of motoneurons innervating the soleus muscle of non-obese rats with type 2 diabetes. In addition, this study examined the effects of mild hyperbaric oxygen at 1.25 atmospheres absolute with 36 % oxygen for 10 weeks on the oxidative capacity of motoneurons innervating the soleus muscle because mild hyperbaric oxygen improves the decreased oxidative capacity of the soleus muscle in non-obese rats with type 2 diabetes. Spinal motoneurons innervating the soleus muscle were identified using nuclear yellow, a retrograde fluorescent neuronal tracer. Thereafter, the cell body sizes and succinate dehydrogenase activity of identified motoneurons were analyzed. Decreased succinate dehydrogenase activity of small-sized alpha motoneurons innervating the soleus muscle was observed in rats with type 2 diabetes. The decreased succinate dehydrogenase activity of these motoneurons was improved by mild hyperbaric oxygen. Therefore, we concluded that rats with type 2 diabetes have decreased oxidative capacity in motoneurons innervating the soleus muscle and this decreased oxidative capacity is improved by mild hyperbaric oxygen.     

Effects of iron deficiency and training on mitochondrial enzymes in skeletal muscle

We measured mitochondrial enzyme activities in skeletal muscle under conditions of iron deficiency and endurance training to assess the effects of these interventions on the contents and proportions of non-iron-containing and iron-dependent enzymes and proteins. Male Sprague-Dawley rats, 21 days of age, received a diet containing either 6 (iron deficient) or 50 mg iron/kg diet (iron sufficient). At 35 days of age animals were subdivided into sedentary and endurance training groups (running at 0.7 mph, 0% grade, 45 min/day, 6 days/wk). By 70 days of age, iron deficiency had decreased gastrocnemius muscle cytochrome c by 62% in sedentary animals. In contrast, the activities of tricarboxylic acid cycle enzymes were increased, remained unchanged or were slightly decreased, indicating that iron deficiency markedly altered mitochondrial composition. Endurance training increased cytochrome c (35%), tricarboxylic acid cycle enzymes (approximately 15%), and manganese superoxide dismutase (33%) in iron-deficient rats, whereas the same exercise regimen had no effect on the skeletal muscle of iron-sufficient animals. The interactive effect of dietary iron deficiency and mild exercise on mitochondrial enzymes suggests that adaptation to a training stimulus is, to some extent, geared to the relationship between the energy demand of exercise and the capacity for O2 transport and utilization.     

Levels of plasma insulin, leptin and adiponectin, and activities of key enzymes in carbohydrate metabolism in skeletal muscle and liver in fasted ICR mice fed dietary n-3 polyunsaturated fatty acids

The aim of this study was to clarify the mechanisms related to plasma glucose concentration in mice fed a diet rich in n-3 polyunsaturated fatty acids (n-3 PUFAs). Male Crlj:CD-1 (ICR) mice were fed experimental diets containing 6% lard (LD), 6% fish oil (FO) or 4.1% lard plus 1.5% docosahexaenoic acid ethyl ester and 0.4% eicosapentaenoic acid ethyl ester (DE) for 12 weeks. There were no marked differences in plasma glucose and insulin concentration changes on glucose tolerance test between the three dietary groups. At the end of the feeding trial, plasma glucose concentration was significantly lower in fasted mice in the FO group than in those in the LD group (P<.005). Plasma adiponectin concentration was significantly higher in the FO group than in the LD group (P<.05). Hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and glycerophosphate dehydrogenase activities in skeletal muscle tended to be lower in the FO group than in the LD group, while there were no differences in glucokinase and phosphofructokinase activities in liver between the three dietary groups. However, hepatic glycerophosphate dehydrogenase activity was 53-fold and 4.2-fold higher in the FO group than in the LD and DE groups, respectively (P<.0005 and P<.05, respectively). These results suggest that the reduction in plasma glucose concentration in mice fed n-3 PUFAs is mainly caused by acceleration of glucose uptake and glycerol synthesis in the liver rather than in the skeletal muscle.     

Splice mutation in the iron-sulfur cluster scaffold protein ISCU causes myopathy with exercise intolerance

A myopathy with severe exercise intolerance and myoglobinuria has been described in patients from northern Sweden, with associated deficiencies of succinate dehydrogenase and aconitase in skeletal muscle. We identified the gene for the iron-sulfur cluster scaffold protein ISCU as a candidate within a region of shared homozygosity among patients with this disease. We found a single mutation in ISCU that likely strengthens a weak splice acceptor site, with consequent exon retention. A marked reduction of ISCU mRNA and mitochondrial ISCU protein in patient muscle was associated with a decrease in the iron regulatory protein IRP1 and intracellular iron overload in skeletal muscle, consistent with a muscle-specific alteration of iron homeostasis in this disease. ISCU interacts with the Friedreich ataxia gene product frataxin in iron-sulfur cluster biosynthesis. Our results therefore extend the range of known human diseases that are caused by defects in iron-sulfur cluster biogenesis.     

Activity-induced adaptations in skeletal muscles of iron-deficient rabbits

The purpose of this study was to determine whether severe iron deficiency alters the adaptive response of skeletal muscle fibers to a sustained increase in tonic contractile activity. Seven weanling rabbits consumed a low iron diet and underwent phlebotomy twice weekly for 6 mo, resulting in severe anemia (mean Hb 5.5 g/dl). Compared with control animals, tibialis anterior skeletal muscles of iron-deficient animals exhibited reduced concentrations of cytochrome c (4.4 +/- 0.7 vs. 8.6 +/- 0.7 nmol/g tissue; P less than 0.01), and reduced activities of citrate synthase (83 +/- 10 vs. 133 +/- 13 mU/mg protein; P less than 0.01) and cytochrome-c oxidase (2.2 +/- 0.2 vs. 3.6 +/- 0.5 U/mg protein; P less than 0.05). In these muscles mitochondria were swollen and displayed deformed cristae. Less severe biochemical abnormalities were observed in cardiac and soleus skeletal muscles. Ten days of continuous electrical stimulation of the motor nerve supplying anterior compartment muscles of iron-deficient rabbits increased expression of mitochondrial proteins: cytochrome c was increased to 154% of control levels (P less than 0.05), and cytochrome-c oxidase and citrate synthase activities were increased to 199 and 272% of control levels, respectively (P less than 0.005). In addition, electrical pacing increased the fractional volume of mitochondria observed by electron microscopy and reduced the activity of aldolase A by 28% (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat

Summary Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (R_T). The bag₂ intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SDH and GPD activities. Bag₁ intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after R_T acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after R_T acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and R_T acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

The effects of resistance exercise and post-exercise meal timing on the iron status in iron-deficient rats

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin in iron-deficient rats. Post-exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post-exercise nutrition timing on hemoglobin synthesis are unclear. The current study investigated the effect of post-exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ-aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the hemoglobin concentration in iron-deficient rats. Male 4-week-old Sprague-Dawley rats were fed an iron-deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/day, 3 days/week) for 3 weeks. The rats were divided into a group fed a post-exercise meal early after exercise (E) or a group fed the meal 4 h after exercise (L). A single bout of exercise performed after the 3-week training period increased the bone marrow ALAD activity, plasma iron concentration, and transferrin saturation. Although the plasma iron concentration and transferrin saturation were lower in the E group than the L group after a single bout of exercise, the basal hematocrit, hemoglobin, and TIBC after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post-exercise meal timing has no effect on the hemoglobin concentration in iron-deficient rats.     

Changes in blood glucose, blood and muscle lactate,and plasma lactic dehydrogenase levels in the pigeon on acute exposure to cold.

Adult pigeons were subjected to acute cold exposure (-25 degrees C; 30 min) after which the levels of blood glucose, blood and muscle lactate and plasma lactic dehydrogenase were measured. Partially defeathered (dorsum and pectoral regions) birds, following exposure to cold, showed marked reduction in blood glucose and blood and muscle (pectoralis) lactate. Fully plumed birds, in contrast, showed no significant reduction in body temperature or blood glucose and only moderately reduced lactate levels indicating the effectiveness of the insulative feather coat in maintaining thermal and metabolic homeostasis. The partially-defeathered pigeons exposed to cold showed a two-to-three-fold increase in plasma lactic dehydrogenase activity, which may reflect a molecular adaptation in their calorigenic response to cold.     

Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance

Background

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.

Methodology/Principal Findings

Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.

Conclusions/Significance

The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.     

The activity of tissue enzymes in iron-deficient rat and man: an overview

The effects of iron deficiency in rat and/or man on iron-containing enzymes of different tissues is reviewed. Iron deficiency results in a decrease of skeletal muscle iron containing proteins e.g. myoglobin, cytochromes c, a + a3, and alpha-glycerophosphate oxidase. Iron deficiency produces a reduction in the activity of several respiratory enzymes in the mitochondrial fraction of cardiac muscle, particularly: NADH cytochrome c reductase, succinic cytochrome c reductase, succinic dehydrogenase and NADH ferricyanide oxidoreductase. The effects of iron deficiency on brain tissue is emphasized with respect to cytochromes, monoaminoxidase and amino acids metabolism. Host defence to infection (controversial data), decrease in body temperature, alteration of DNA synthesis, collagen and lipid metabolism, liver and gastrointestinal mucous cytochromes activity perturbations are discussed.     

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms.     

Hepatic adaptations to iron deficiency and exercise training.

Brooks et al. [Am. J. Physiol. 253 (Endocrinol. Metab. 16): E461-E466, 1987] demonstrated an elevated gluconeogenic rate in resting iron-deficient rats. Because physical exercise also imposes demand on this hepatic function, we hypothesized that exercise training superimposed on iron deficiency would augment the hepatic capacity for amino acid transamination/deamination and pyruvate carboxylation. Sprague-Dawley rats (n = 32) were obtained at weaning (21 days of age) and randomly assigned to iron-sufficient (dietary iron = 60 mg iron/kg diet) or iron-deficient (3 mg iron/kg) dietary groups. Dietary groups were subdivided into sedentary and trained subgroups. Treadmill training was 4 wk in duration, 6 days/wk, 1 h/day, 0% grade. Treadmill speed was initially 26.8 m/min and was decreased to 14.3 m/min over the 4-wk training period. The mild exercise-training regimen did not affect any measured variable in iron-sufficient rats. In contrast, in iron-deficient animals, training increased endurance capacity threefold and reduced blood lactate and the lactate-to-alanine ratio during submaximal exercise by 34 and 27%, respectively. The mitochondrial oxidative capacity of gastrocnemius muscle was increased 46% by training. However, the oxidative capacity of liver was not affected by either iron deficiency or training. Maximal rates of pyruvate carboxylation and glutamine metabolism by isolated liver mitochondria were also evaluated. Iron deficiency and training interacted to increase pyruvate carboxylation by intact mitochondria. Glutamine metabolism was increased roughly threefold by iron deficiency alone, and training amplified this effect to a ninefold increase over iron-sufficient animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical studies of fibroblasts from patients with Menkes kinky hair disease and Wilson's disease

Summary Cultured fibroblasts from Menkes kinky hair disease patients showed markedly reduced succinate dehydrogenase and amine oxidase activities. Cytochrome oxidase activity, however, was greatly reduced in some cells and almost normal in others. Cultured fibroblasts from patients with Wilson's disease showed moderately reduced succinate dehydrogenase and cytochrome oxidase activities. Amine oxidase activity was only slightly reduced when compared to that of normal. These results indicated that the histochemical phenotype observed in fibroblasts from patients with Menkes kinky hair disease and Wilson's disease were distinctly different from each other and from normal fibroblasts.     

Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

Enzyme activities in muscle and connective tissue of M. Vastus lateralis in habitually training and sedentary 33 to 70-year-old men.

A cross-sectional study was carried out to examine the activities of certain enzymes representing aerobic and anaerobic energy metabolism as well as the biosynthesis of collagen of M. vastus lateralis in 23 male endurance athletes in habitual training, aged 33 to 70 years. 23 sedentary healthy men of corresponding ages were selected for the control group. The mean maximal oxygen uptake of the trained subjects was 53.6 ml-kg--1. min--1 and that of the control subjects 36.3 ml-kg--1. min--1. As compared to the control group the trained subjects had significantly higher values in the muscle malate dehydrogenase, succinate dehydrogenase and prolyl hydroxylase activities, whereas the opposite was true in the activity of lactate dehydrogenase. In hexokinase and creatine phosphokinase no marked differences between the groups were observed. The results showed that endurance training leads to increased activities of oxidative enzymes in the skeletal muscle. The adaptation changes were also observed in old men. The increased activity of prolyl hydroxylase may reflect the general enzymatic adaptation to physical training. A possibility exists that the turnover of muscle collagen in endurance athelets is continuously faster than that in sedentary men of corresponding ages.     

Changes induced by Fe deficiency and Fe resupply in the organic acid metabolism of sugar beet (Beta vulgaris) leaves

The effects of iron deficiency and iron resupply on the metabolism of leaf organic acids have been investigated in hydroponically grown sugar beet. Organic acid concentrations and activities in leaf extracts of several enzymes related to organic acid metabolism were measured. Enzymes assayed included phosphoenol pyruvate carboxylase (PEPC; EC 4.1.1.31), different Krebs cycle enzymes: malate dehydrogenase (MDH; EC 1.1.1.37), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2), citrate synthase (CS; EC 4.1.3.7) and isocitrate dehydrogenase (ICDH; EC 1.1.1.42), glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and two enzymes related to anaerobic metabolism (lactate dehydrogenase [LDH]; EC 1.1.1.27, and pyruvate decarboxylase [PDC]; EC 4.1.1.1). Iron concentration in leaves was severely decreased by iron deficiency. Iron resupply caused an increase in iron concentrations, reaching levels similar to the controls in 96 h. Iron deficiency induced a 2.3-fold (from 16 to 37 mmol m⁻²) increase in leaf total organic acid concentration. Organic anion concentrations were still 4-fold higher than the controls 24 h after resupply and decreased to values similar to those found in the controls after 96 h. All measured enzymes had increased activities in extracts of iron-deficient leaves when compared to the controls and generally decreased to control values 24 h after iron addition. These data provide evidence that organic acid accumulation in iron-deficient leaves is likely not due to an enhancement in leaf carbon fixation. Instead, this accumulation could be associated with organic acid export from the roots to the leaves via xylem.