Defective Electron Transport Research Articles

Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport

Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper–zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction. Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV. Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II + III, III, and IV and corresponding decreases in hemes b, c-c 1, and a-a 3 content compared to mitochondria from wild type human SOD1-expressing yeast. Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a 3 content. These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.

Determination of mitochondrial function and site-specific defects in electron transport in duodenal mitochondria in broilers with low and high feed efficiency

Duodenal mitochondria were isolated from broiler breeder males with high (0.79+/-0.01, n = 9) and low (0.63+/-0.02, n = 9) feed efficiency (FE) to assess relationships of FE with duodenal mitochondrial function and site-specific defects in electron transport. Sequential additions of adenosine diphosphate (ADP) resulted in 1) higher respiratory control ratio (RCR; an index of respiratory chain coupling) in high FE mitochondria provided succinate, and 2) higher ADP to oxygen ratio (ADP:O; an index of oxidative phosphorylation) in low FE mitochondria provided NADH-linked substrates (malate, pyruvate, or both). Basal electron leak, measured as H2 O2 production, was greater in low FE mitochondria provided succinate (P = 0.08) or NADH-linked substrates. As H2 O2 levels were elevated in low FE compared with high FE mitochondria by complex I (P+/-0.07) and complex II inhibition, the higher basal electron leak in low FE mitochondria was apparently due to site-specific defects in electron transport at complexes I and II. Elevations in H2 O2 above basal levels indicated that high FE mitochondria may also exhibit electron transport defects at complexes I and III. Despite an ability to produce adenosine triphosphate (ATP) that was equal or superior to that demonstrated in high FE duodenal mitochondria, low FE mitochondria exhibited a greater inherent degree of electron leak. The results provide insight into the role that duodenal mitochondria play in the phenotypic expression of FE in broilers.

Role of Mitochondria in the Phenotypic Expression of Feed Efficiency

Recent investigations were conducted on mitochondria obtained from broilers with low and high feed efficiency (FE) within the same genetic line that were fed the same diet, thereby allowing relationships between mitochondrial function and the phenotypic expression of FE to be clearly established. In these studies, breast muscle, liver and duodenal mitochondria obtained from broilers with high FE exhibited a more tightly coupled respiratory chain along with lower electron leak and reactive oxygen species (ROS) production compared with broilers with low FE. Elevated electron leak in low FE was due to site-specific defects in electron transport at Complex I, III, or both of the respiratory chains in breast and leg muscles and at Complex I, II, or III (depending on energy substrate) in duodenal mitochondria. Increased ROS production was presumably responsible for increased protein oxidation in low FE muscle mitochondria. Of 14 respiratory chain proteins investigated in breast muscle, the expression of 4 (3 in Complex III and 1 in Complex IV) was higher in low FE mitochondria. Interestingly, low FE was associated with a general suppression in respiratory chain complex activities (I, II, III, and IV) in breast muscle and liver. Elucidation of the mechanisms responsible for lower ROS production and tighter coupling of the respiratory chain in high FE mitochondria will greatly facilitate our understanding of the cellular basis for the phenotypic expression of FE.

Is defective electron transport at the hub of aging?

The bulwark of the mitochondrial theory of aging is that a defective respiratory chain initiates the death cascade. The increased production of superoxide is suggested to result in progressive oxidant damage to cellular components and particularly to mtDNA that encodes subunits assembled in respiratory complexes. Earlier studies of respiration in muscle mitochondria obtained from large cohorts of patients supported this notion by showing that either singly or in combinations, the respiratory complexes exhibited decreased activity in the elderly. The following critique of the most cited publications over the past decade points out the systematic errors that put earlier work at odds with recent findings. These later investigations indicate that aging has no overt effect on either the electron transport system or oxidative phosphorylation.

Ischemia–reperfusion injury in the aged heart: role of mitochondria

The aged heart sustains greater injury during ischemia and reperfusion compared to the adult heart. Aging decreases oxidative phosphorylation and the activity of complexes III and IV only in interfibrillar mitochondria (IFM) that reside among the myofibrils, whereas subsarcolemmal mitochondria (SSM), located beneath the plasma membrane, remain unaltered. The peptide subunit composition of complexes III and IV is intact in aging. The aging defect in complex IV is in the inner membrane lipid environment. The defect in complex III is within the ubiquinol binding site of the cytochrome b subunit. Following ischemia, in the aged heart both SSM and IFM sustain additional decreases in complex III and complex IV activity. In contrast to the aging defect, with ischemia the subunits of complex IV appear to be damaged. Ischemia inactivates the iron–sulfur peptide subunit in complex III. Mitochondria are the major source of the reactive oxygen species that are generated during myocardial ischemia. Complex III is the major site of mitochondrial oxyradical production during ischemia in the adult heart. The role of complex III in the oxidative damage sustained by the aged heart during ischemia, as well as the potential contribution of aging defects in electron transport to ischemic damage in the aged heart, deserves further study. We propose that following ischemic damage to the electron transport chain, the production and release of reactive oxygen species increases from mitochondria in the aged heart, leading to additional damage during reperfusion.

Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers ( Gallus domesticus)

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b 562). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.

Mechanisms of cell death in hypoxia/reoxygenation injury.

Investigation of death pathways during cell injury in vivo caused by ischemia and reperfusion is of clinical importance, but technically difficult. Heterogeneity of cell types, differences between organ systems, diversity of death paradigms and exacerbation of tissue damage caused by inflammation are only some of the variables that need to be taken into account. With respect to the identification of necrosis and apoptosis in affected organs, technical issues related to preparation artifacts, occurrence of internucleosomal DNA cleavage in necrotic as well as apoptotic cells and other overlaps in death pathways bear consideration. In that caspase independent as well as caspase dependent processes cause cell death and that caspase inhibitors can act as anti-inflammatory agents, evaluation of ischemic death mechanisms in parenchymal cells needs to be performed with caution. When the effects of inflammation are removed by appropriate in vitro studies using purified or cultured cells, several mitochondrial factors that lead to cell death can be studied. Substantial evidence exists for the participation of electron transport defects, mitochondrial permeability transitions (MPT) and release of cytochrome c from mitochondria, effected by pro-apoptotic proteins such as Bax. The anti-apoptotic protein Bcl-2 exerts an overriding protective role in this type of injury by preserving mitochondrial structure and function. In contrast, caspase inhibitors cannot offer long-term protection to ischemically injured parenchymal cells regardless of how effectively they can inhibit apoptotic events, if the cells have suffered permanent mitochondrial damage impairing respiration.

Defective repair of oxidative damage in mitochondrial DNA in Down's syndrome

Recent evidence indicates that oxidative DNA damage may be a major cause of aging. One of the more sensitive targets is the mitochondrial genome which is 10 times more susceptible to mutation than is the nuclear genome. A number of age-related neuromuscular degenerative diseases also have been associated with mutations in mitochondrial DNA (mtDNA), and progressive accumulation of oxidative damage in mtDNA from neuronal tissues over time has been shown. In support of the notion that oxidative stress leads to aging is the finding in Down's syndrome (DS), which is characterized by premature aging, that there is enhanced oxidative stress resulting from the aberrant expression of CuZn superoxide dismutase (CuZn SOD). On the basis of these observations, we hypothesized that there may be defective repair of oxidative damage in mtDNA which would ultimately lead to defective electron transport and concomitant enhanced production of reactive oxygen species (ROS). This effect would heighten the oxidative burden in the cell and accelerate the development of phenotypes associated with aging. To evaluate repair of oxidative damage in mtDNA, fibroblasts from several DS patients were treated with the reactive oxygen generator menadione. Oxidative damage was assessed at 0, 2, and 6 h after exposure using a Southern-blot technique and a mtDNA specific probe. The results of these studies show that DS cells are impaired in their ability to repair oxidative damage to mtDNA compared to age-matched control cells. Therefore, this data supports the possibility that increased production of ROS from mitochondria plays a crucial role in the development of aging phenotypes.

Disorders of the mitochondria.

Recent advances in our understanding of the structure and function of mitochondria have led to the recognition that inherited and acquired mitochondrial dysfunction may be responsible for diseases affecting the liver and other organ systems. Mitochondrial health may also determine hepatocyte survival in other hepatic disorders not directly related to the mitochondrion. Primary mitochondrial hepatopathies are conditions in which there are inherited defects in structure or function of the mitochondria, most of which involve the respiratory chain and oxidative phosphorylation, fatty acid oxidation, the urea cycle, and other pathways confined to mitochondria. Maternally inherited mutations or deletions of the mitochondrial genome, or putative nuclear gene mutations encoding electron transport proteins, cause defective electron transport, oxidative stress, impaired oxidative phosphorylation, and other metabolic derangements that lead to hepatic failure or chronic liver dysfunction in affected children. The mitochondrial DNA (mtDNA) depletion syndrome, which similarly leads to liver failure and neurologic abnormalities, is caused by a putative nuclear gene that controls mtDNA replication or stability. Other proven or suspected primary mitochondrial hepatopathies include Pearson's marrow-pancreas syndrome, Alpers disease, mitochondrial neurogastrointestinal encephalomyopathy syndrome, and Navajo neuropathy. Secondary mitochondrial hepatopathies are conditions in which the mitochondria are major targets during liver injury from another cause, such as metal overload, certain drugs and toxins, alcoholic liver injury, and conditions of oxidant stress. Diagnosis of mitochondrial dysfunction may be difficult with currently available tools, however, elevated blood lactate: pyruvate ratios or arterial ketone body ratios with characteristic liver histology are initial tests. Measuring respiratory chain enzyme activities, mtDNA levels, and searching for mtDNA mutations and deletions are more specific tests. Treatment of these disorders is currently empirical, involving agents that may improve the redox status of mitochondria, promote electron flow, or act as mitochondrial antioxidants. Liver transplantation has occasionally been successful in patients who lack other systemic involvement.

Diet change in the management of metabolic encephalomyopathies.

Dietary treatment of mitochondrial disease caused by oxidative metabolic defects has traditionally been limited to the use of ketogenic diets for the treatment of pyruvate dehydrogenase (PDH) deficiency. However, defects in mitochondrial function resulting in a failure of oxidative metabolism can result in a block in the utilization of various food substrates and dietary manipulation might be beneficial. Certain predictions might be made based on the site of particular enzymatic blocks. Some disorders primarily block a particular substrate pathway, such as PDH deficiency which predominantly affects the utilization of carbohydrates and acyl-CoA dehydrogenase deficiencies which predominantly affect fat metabolism. Defects of the terminal electron transport chain, ubiquinone to cytochrome oxidase (COX), affect the ‘final common pathway’ for energy production and might be expected to impair the utilization of all food substrates. In complex I deficiency utilization of carbohydrates which generate NADH, might be expected to be impaired. The β-oxidation of fats produces NADH, ETFred and acetyl-CoA. Reducing equivalents derived from β-oxidation enter the electron transport chain at both site 1 (complex I) and site 2 (ubiquinone). Electron transport defects involving complex I may theoretically benefit from the use of high fat diets as reducing equivalents from ETFred can be introduced through the ETF/ubiquinone pathway from the metabolism of fatty acids. In patients whose lactic acid production is increased by carbohydrate intake, fructose, which is rapidly metabolized to pyruvate, might be less well tolerated than glucose as a food source.

1341 Cell death of adult canary forebrain induced by heavy ion irradiation

We tested quercetin, a dietary bioflavonoid with potent free radical scavenging action and antioxidant activity, for its neuroprotective effects in rotenone-induced hemi-parkinsonian rats. Rats were infused unilaterally with rotenone into the substantia nigra, and quercetin (25–75 mg/kg, i.p.) was administered at 12-h intervals for 4 days, and analyzed on the 5th day. Amphetamine- or apomorphine-induced unilateral rotations were significantly reduced in quercetin-treated rats, when analyzed on 14th or 16th day post-surgery, respectively. Quercetin possessed potent hydroxyl radical scavenging action in a cells-free, Fenton-like reaction in test tubes, and in isolated mitochondria when measured by salicylate hydroxylation method. We observed dose-dependent attenuation of the rotenone-induced loss in striatal dopamine, and nigral oxidized and reduced glutathione, as well as the increases in endogenous antioxidant enzymes (catalase and superoxide dismutase) activities supporting the notion that quercetin-effect is mediated via its powerful hydroxyl radicals-scavenging and antioxidant actions. Quercetin’s dose-dependent ability to up-regulate mitochondrial complex-I activity, as evidenced by NADH-oxidation, and as seen in blue native-polyacrylamide gel electrophoresis (PAGE) staining in both the contra- and ipsi-lateral nigra suggests the containment of reactive oxygen production at the mitochondrial level. Rotenone-induced induction of NADH-diaphorase activity in the nigral neurons, and its attenuation by quercetin pointed to the possible involvement of nitric oxide too. Reversal of neuronal death induced by rotenone as observed by increased tyrosine hydroxylase-positive cells and decreased TdT-mediated dUTP nick end-labeling (TUNEL) staining in the substantia nigra confirmed the potential of quercetin to revamp dopaminergic cells following oxidative stress mediated programmed cell death and neuronal demise. The present study strongly implicates quercetin’s potential ability to repair mitochondrial electron transport defects and to up-regulate its function as the basis of neuroprotection observed in a mitochondrial neurotoxin-induced Parkinsonism.

Yeast lacking superoxide dismutase. Isolation of genetic suppressors.

Null mutants of superoxide dismutase (SOD) in Saccharomyces cerevisiae are associated with a number of biochemical defects. In addition to being hypersensitive to oxygen toxicity, strains containing deletions in both the SOD1 (encoding Cu/Zn-SOD) and SOD2 (encoding Mn-SOD) genes are defective in sporulation, are associated with a high mutation rate, and are unable to biosynthesize lysine and methionine. The sod-linked defect in lysine metabolism was explored in detail and was found to occur at an early step in lysine biosynthesis, evidently at the level of the alpha-amino adipate transaminase. To better understand the role of SOD in cell metabolism, our laboratory has isolated yeast suppressors that have bypassed the SOD defect ("bsd" strains), that is, S. cerevisiae cells lacking SOD, yet resistant to oxygen toxicity. Two nuclear bsd complementation groups have been identified, and both suppress a variety of biological defects associated with sod1 and sod2 null mutants. These results demonstrate that a single gene mutation can alleviate the requirement for SOD in cell growth. Both bsd complementation groups are unable to utilize many non-fermentable carbon sources, suggesting a possible suppressor-linked defect in electron transport.

Genetic analysis of photosynthesis in Rhodospirillum centenum.

A genetic system has been developed for studying bacterial photosynthesis in the recently described nonsulfur purple photosynthetic bacterium Rhodospirillum centenum. Nonphotosynthetic mutants of R. centenum were obtained by enrichment for spontaneous mutations, by ethyl methanesulfonate mutagenesis coupled to penicillin selection on solid medium, and by Tn5 transposition mutagenesis with an IncP plasmid vector containing a temperature-sensitive origin of replication. In vivo and in vitro characterization of individual strains demonstrated that 38 strains contained mutations that blocked bacteriochlorophyll a biosynthesis at defined steps of the biosynthetic pathway. Collectively, these mutations were shown to block seven of eight steps of the pathway leading from protoporphyrin IX to bacteriochlorophyll a. Three mutants were isolated in which carotenoid biosynthesis was blocked early in the biosynthetic pathway; the mutants also exhibited pleiotropic effects on stability or assembly of the photosynthetic apparatus. Five mutants failed to assemble a functional reaction center complex, and seven mutants contained defects in electron transport as shown by an alteration in cytochromes. In addition, several regulatory mutants were isolated that acquired enhanced repression of bacteriochlorophyll in response to the presence of molecular oxygen. The phenotypes of these mutants are discussed in relation to those of similar mutants of Rhodobacter and other Rhodospirillum species of purple photosynthetic bacteria.

Hepatic mitochondrial oxidative metabolism and lipid peroxidation in experimental hexachlorobenzene-induced porphyria with dietary carbonyl iron overload.

Both human porphyria cutanea tarda and experimental hexachlorobenzene-induced porphyria are associated with hepatic injury and are potentiated by excess hepatic iron. The mechanisms whereby cellular injury occurs and the synergistic role of iron overload are unknown. In the present experiments, we studied hepatic mitochondrial function and lipid peroxidation in rats with hexachlorobenzene-induced porphyria in which iron loading was achieved by dietary carbonyl iron supplementation. Female rats were treated for 8 weeks, receiving a chow diet supplemented with hexachlorobenzene (0.2%, w/w), carbonyl iron (1.0%, w/w) or hexachlorobenzene + iron. Hepatic total porphyrins were increased 100-fold in rats receiving hexachlorobenzene (hexachlorobenzene alone and hexachlorobenzene + Fe), and total hepatic iron was increased approximately 10-fold in rats receiving iron supplementation (Fe alone and hexachlorobenzene + Fe). There was a significant increase in mitochondrial lipid peroxidation in rats treated with hexachlorobenzene alone and hexachlorobenzene + Fe. A significant reduction in mitochondrial respiratory control ratios and in oxidative phosphorylation (ADP/O ratios) using glutamate and succinate as substrates was demonstrated when rats were treated with hexachlorobenzene + iron. The reductions in respiratory control ratios were due to a combination of an inhibitory defect in electron transport as evidenced by an irreversible decrease in State 3 respiration and an uncoupling effect as evidenced by an increase in State 4 respiration. These findings suggest that lipid peroxidation and mitochondrial dysfunction may contribute to the hepatotoxicity seen in hexachlorobenzene-induced porphyria.

Skeletal Muscle Disorders and Associated Factors That Limit Exercise Performance

The study of skeletal muscle disorders is providing potentially important insights into regulatory mechanisms in human exercise and fatigue and information useful for diagnostic and treatment purposes. This review primarily concerned the general metabolic and physiological factors which set upper limits to performance of various types of exercise in patients with a variety of muscle disorders. From the standpoint of exercise performance, skeletal muscle diseases can be classified into three major groups. One group consists of primary disorders of muscle energy metabolism, including defects in muscle carbohydrate and lipid metabolism, disorders of mitochondrial electron transport, and abnormalities of purine nucleotide metabolism. Exercise performance largely reflects the capacity for ATP resynthesis. Oxidative phosphorylation is the dominant quantitative source of energy for ATP resynthesis under most exercise conditions. Consequently, patients with disordered oxidative metabolism (i.e., patients with defects in the availability or utilization of oxidizable substrate, such as those with phosphorylase or PFK deficiency or those with defects in mitochondrial electron transport) typically demonstrate severely impaired exercise performance. Intolerance to sustained exercise and premature fatigability are salient features of muscle oxidative disorders. Maximal oxygen uptake and maximal a-v O2 difference are markedly subnormal related to an attenuated muscle oxygen extraction. Muscle weakness and atrophy are less common. Anaerobic muscle performance is dramatically limited in patients with virtually complete defects of glycogenolysis/glycolysis but appears relatively normal in those with electron transport defects. A second major group of disorders includes patients with decreased muscle mass due to muscle necrosis, atrophy, and replacement of muscle by fat and connective tissue. These disorders are exemplified by the various muscular dystrophies (Duchenne's dystrophy, Becker's dystrophy, LG dystrophy, FSH dystrophy, and myotonic dystrophy) in which exercise performance is severely impaired due to muscle wasting and weakness in spite of largely normal pathways for muscle ATP resynthesis. In muscular dystrophy patients, the degree to which maximal oxygen uptake and anaerobic muscle performance are impaired appears to be a function of the severity of muscle weakness and atrophy. A third group of disorders includes patients with impaired activation of muscle contraction or relaxation. These disorders may be considered in two subcategories. In the first, impaired activation or relaxation of contractile activity is due to intrinsic muscle dysfunction (e.g., diseases associated with myotonia or periodic paralysis). In the second subcategory, there is impaired muscle activation due to a primary abnormality in the central nervous system, motor nerves, or neuromuscular junction.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome b_245 and its Involvement in the Molecular Pathology of Chronic Granulomatous Disease

Cytochrome b-245 is an integral, and probably the terminal, component of the microbicidal oxidase electron transport chain of phagocytic cells. Current knowledge of the biochemistry and cell and molecular biology of this molecule is described. The molecular basis of chronic granulomatous disease, in which defective electron transport down this chain predisposes to infection and impaired digestion by phagocytes, is explained in terms of anomalies of the cytochrome b and related molecules.

31P NMR study of improvement in oxidative phosphorylation by vitamins K3 and C in a patient with a defect in electron transport at complex III in skeletal muscle.

The bioenergetic capacity of skeletal muscle in a 17-year-old patient with a severe defect in complex III of the electron transport chain has been examined by 31P NMR measurements of the molar ratio of phosphocreatine to inorganic phosphate (PCr/Pi). Resting ratios were 1.3-2.5, which can be compared with roughly 8.6 for a young, normal female control at rest. Quantitative evaluation of the activity of oxidative metabolism was afforded by the rate of recovery of PCr/Pi from exercise and was found to be 2.5% of normal. After administration of menadione and ascorbate, we found a 21-fold increase of the recovery rate relative to the pretherapy value, to within 56% of the recovery rate of the young female control. Thus, NMR examinations of skeletal muscle at rest and in recovery from activity document marked improvement to specific drug therapy in the electron transport capabilities and the ATP synthesis rate of a patient with a deficiency in a cytochrome b-containing complex III. Improvements in functional ability, although not as dramatic as biochemical changes, are also apparent.

Abnormal mitochondrial oxidative phosphorylation of ischemic myocardium reversed by Ca 2+-chelating agents

Mitochondria isolated from ischemic and ischemic-reperfused myocardium have been shown to have a defect in electron transport and in energy-linked 45 Ca 2+ uptake. In this study, the influence of Ca 2+ on the efficiency of oxidative phosphorylation by mitochondria isolated from ischemic and ischemic-reperfused myocardium was studied by directly measuring ATP production in the presence and absence of Ca 2+ -chelating agent—EDTA and EGTA. Results demonstrate the following. (a) Mitochondria isolated from ischemic myocardium have a low rate of ATP production, reduced ATP/O ratios and lower net ATP production. These functions are improved by EDTA. (2) Mitochondria from ischemic-reperfused myocardium are totally incapable of phosphorylating ADP in the absence of Ca-chelating agents. Addition of EDTA or EGTA either to the isolation medium or to the incubation medium restores the ability of these mitochondria to phosophorylate all added ADP, although the rate of this phosphorylation remained very slow. (3) The Ca 2+ content of mitochondria from ischemic-reperfused myocardium was higher than the Ca 2+ levels of mitochondria from either normal or ischemic myocardium. These results suggest that phosphorylation of ADP by mitochondria from ischemic and ischemic-reperfused myocardium is inhibited by endogeneous ionic Ca 2+ and that this inhibition can be partially reversed by the addition of Ca 2+ -chelating agents.