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Y H Wei

Publications and source records attributed to Y H Wei.

At least 37 records · Page 2Linked to original sources
PubMed2000

Large-scale mitochondrial DNA deletions in skeletal muscle of patients with end-stage renal disease.

End-stage renal disease (ESRD) is associated with enhanced oxidative stress. This disease state provides a unique system for investigating the deleterious effect of exogenous sources of free radicals and reactive oxygen species (ROS) on mitochondrial DNA (mtDNA). To test the hypothesis that uremic milieu might cause more severe damage to mtDNA, we investigated the prevalence and abundance of mtDNA deletions in the skeletal muscles of ESRD patients. The results showed that the frequencies of occurrence of the 4977 bp and 7436 bp deletions of mtDNA in the muscle tissues of the older ESRD patients were higher than those of the younger patients. The frequency of occurrence of the 4977 bp-deleted mtDNA in the muscle was 33.3% for the patients in the age group of < 40 years, 66.6% in the 41-60-year-old group, 100% in the 61-80-year-old group, and 100% in patients >80 years of age, respectively. Only 22% of the normal aged controls carried the 4977 bp mtDNA deletion, whereas 77% (17/22) of the ESRD patients exhibited the mtDNA deletion. Using a semiquantitative PCR method, we determined the proportion of the 4977 bp-deleted mtDNA from the muscles that had been confirmed to harbor the deletion. We found that the proportions of the 4977 bp-deleted mtDNA in the muscle were significantly higher than those of the aged matched controls. Using long-range PCR techniques, a distinctive array of mtDNA deletions was demonstrated in the muscle of uremic patients. In summary, we found diverse and multiple mtDNA deletions in the skeletal muscles of ESRD patients. These deletions are more prevalent and abundant in ESRD patients than those found in normal populations. Accumulation of uremic toxins and impaired free radical scavenging systems may be responsible for the increased oxidative stress in ESRD patients. Such stress may result in oxidative damage and aging-associated mutation of the mitochondrial genome.

Adult↗
PubMed2000

Autoantibody against oxidized low-density lipoproteins may be enhanced by cigarette smoking.

A total of 59 healthy male subjects (32 smokers and 27 nonsmokers) who had no reported systemic disease and did not take alcohol and vitamin supplementation were included. The levels of autoantibody to oxidized low-density lipoproteins (ox-LDL) in smokers and age-matched nonsmokers were compared. The plasma levels of antioxidants that can affect the formation of ox-LDL were also measured, and correlation analyses between anti ox-LDL IgG and plasma antioxidants, controlling for age and body mass index (BMI), were performed. Plasma alpha-tocopherol and uric acid concentrations of nonsmokers (2.78+/-1.09 microg/mg total lipid and 6.96+/-1.69 mg/dl, respectively) were significantly higher than those of smokers (1.68+/-0.48 microg/mg total lipid and 6.15+/-1.14 mg/dl, respectively) (P<0.05). Although plasma ascorbate and retinol levels were not significantly different between smokers and nonsmokers, smokers older than 45 years old had significantly lower plasma ascorbate levels (0.32+/-0.17 mg/dl) than age-matched nonsmokers (0. 53+/-0.14 mg/dl) (P=0.036). Higher level of plasma anti ox-LDL IgG was noted in the group of smokers compared with nonsmokers (515+/-409 mU/ml vs. 407+/-268 mU/ml, respectively) under the statistic method of Chi-Square test (P=0.049). A significant negative correlation was found between plasma anti ox-LDL IgG and alpha-tocopherol in the combined population as well as in the smoker group (r=-0.26, p=0.047; r=-0.48, p=0.006; respectively). However, there was no correlation between plasma anti ox-LDL IgG and the levels of other antioxidants. These results suggest that reduced concentrations of alpha-tocopherol are associated with cigarette smoking. The significantly negative correlation between plasma anti ox-LDL IgG and alpha-tocopherol in the entire study population as well as in the smoker group suggests that plasma alpha-tocopherol may be partially effective if not totally at protecting LDL from oxidative damage caused by cigarette smoking and dietary supplementation with alpha-tocopherol may provide a protective effect against LDL oxidation, especially in smokers.

Adult↗
PubMed2000

Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells.

Mitochondrial respiratory function is impaired in the target tissues of patients with mitochondrial diseases and declines with age in various human tissues. It is generally accepted that respiratory-chain defects result in enhanced production of reactive oxygen species and free radicals in mitochondria. Recently, we have demonstrated that the copy number of mitochondrial DNA (mtDNA) is increased in the lung tissues of elderly human subjects. The mtDNA copy number was suggested to be increased by a feedback mechanism that compensates for defects in mitochondria harbouring mutated mtDNA and a defective respiratory system. However, the detailed mechanism remains unclear. In this study, we treated a human lung fibroblast cell line, MRC-5, with H(2)O(2) at concentrations of 90-360 microM. After the treatment for 24-72 h, we found that cells were arrested at G(0) and G(1) phases but that mitochondrial mass and mtDNA content were significantly increased in a concentration- and time-dependent manner. Moreover, the oxidative stress induced by buthionine sulphoximine was also found to cause an increase in mitochondrial mass of the treated cells. Increased uptake of a vital mitochondrial dye Rhodamine 123 and enhanced tetrazolium [MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction revealed that the mitochondria increased by H(2)O(2) treatment were functional. In addition, the increase in the mitochondrial mass was also observed in cell-cycle-arrested cells induced by mimosine, lovastatin and genistein. Taken together, these findings suggest that the increase in mitochondrial mass and mtDNA content are the early molecular events of human cells in response to endogenous or exogenous oxidative stress through cell-cycle arrest.

Cell Cycle↗
PubMed2000

Mitochondrial DNA deletion of the human detrusor after partial bladder outlet obstruction-correlation with urodynamic analysis.

OBJECTIVES: To investigate mitochondrial DNA (mtDNA) mutations in human detrusor after partial bladder outlet obstruction (BOO) and correlate the findings with the results of urodynamic studies. METHODS: Sixty-two male patients with and without BOO were recruited and assessed by the International Prostate Symptom Score, a quality-of-life assessment index, and sonography. The severity of partial BOO was determined by pressure-flow study with an International Continence Society (ICS) nomogram. Random detrusor biopsies obtained cystoscopically were analyzed by polymerase chain reaction (PCR) techniques to detect possible mtDNA deletions. Primer-shift PCR and DNA sequencing were then performed to characterize specific mtDNA deletions. A semiquantitative PCR method was used to determine the proportion of the deleted mtDNA in detrusor. Finally, the mtDNA deletion and the urodynamic results were compared statistically. RESULTS: A 4977-bp mtDNA deletion was identified in the human detrusor. Its incidence and proportion were found to increase after partial BOO (P = 0.005 and 0.012, respectively). The incidence of the mtDNA deletion was 4.2% (1 of 24) in the unobstructed group, 27.8% (5 of 18) in the equivocal group, and 40% (8 of 20) in the obstructed group. The mean proportion of the 4977-bp deleted mtDNA was 23.7 and 12.7 times higher in the obstructed and equivocal groups, respectively, compared with that of the unobstructed group. CONCLUSIONS: We found mtDNA with the 4977-bp deletion in human detrusor and an increase of this deletion after partial BOO. This molecular change might account for the previous observations of mitochondrial functional impairment and voiding dysfunction after partial BOO.

Aged↗
PubMed2000

Effect of vitamin E-bonded membrane on the 8-hydroxy 2'-deoxyguanosine level in leukocyte DNA of hemodialysis patients.

BACKGROUND: 8-Hydroxy 2'-deoxyguanosine (8-OHdG) of leukocyte DNA has been identified as a surrogate marker of oxidative stress in chronic hemodialysis (HD) patients. In this study, we focused on the determinants of the 8-OHdG level in leukocyte DNA of HD patients. We further investigated the influence of vitamin E-modified, regenerated cellulose (CL-E) membrane on the oxidative DNA damage, intracellular reactive oxygen species (ROS) production of granulocytes, and plasma alpha-tocopherol concentration. METHODS: 8-OHdG content in cellular DNA of leukocytes was measured by a high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method. Intracellular production of ROS, H2O2 and O2-. were analyzed by flow cytometry in leukocytes with and without phorbol-12-myristate-13-acetate (PMA) stimulation before dialysis, as well as at 15 and 30 minutes of dialysis. Plasma alpha-tocopherol concentration was measured by a HPLC method, and the value of alpha-tocopherol was corrected by total blood lipid concentration. RESULTS: In the prospective cross sectional study, the mean 8-OHdG level in leukocyte DNA was equally lower in the patients of the CL-E, polymethylmethacrylate (PMMA), and polysulfone (PS) groups as compared with the cellulosic group (ANOVA, P < 0.001). The leukocyte 8-OHdG level correlated negatively with plasma alpha-tocopherol and blood lipid-adjusted plasma alpha-tocopherol, but correlated positively with serum iron and percentage of transferrin saturation. Forward stepwise multiple regression showed that dialysis membrane type, serum iron, and blood lipid-adjusted plasma alpha-tocopherol were the independent determinants of the leukocyte 8-OHdG level in HD patients. Like synthetic membranes, granulocyte ROS production was less augmented during dialysis with the CL-E membrane as compared with the cellulose membrane. Exposure to cellulose membrane impaired intracellular ROS production of granulocytes in response to PMA challenge, whereas the CL-E and synthetic membranes improved the granulocyte responsiveness to PMA. In the longitudinal cross-over study, the 8-OHdG level significantly decreased, and blood lipid-adjusted plasma alpha-tocopherol increased after switching the cellulose membrane to CL-E or synthetic membrane for eight weeks. In contrast, the 8-OHdG level dramatically rose, and blood lipid-adjusted plasma alpha-tocopherol declined after shift of CL-E or synthetic membrane to the cellulose membrane. CONCLUSIONS: CL-E membrane exhibited biocompatible and bioactive characteristics. Like synthetic membranes, treatment with a CL-E dialyzer effectively reduced the 8-OHdG content in leukocyte DNA, suppressed intracellular ROS production of granulocytes, and preserved the plasma level of vitamin E. It could further improve granulocyte responsiveness to a PMA challenge. Reduced DNA damage and improved immune function of leukocytes may reduce the cancer and infection risks in chronic HD patients.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

8-hydroxy-2'-deoxyguanosine of leukocyte DNA as a marker of oxidative stress in chronic hemodialysis patients.

In contrast to proteins and lipids, oxidative damage to DNA has not been well studied in patients undergoing hemodialysis (HD). We hypothesized that phagocytes are activated after blood-membrane contact during HD, and oxidants from metabolic activation can damage leukocyte DNA. To test this hypothesis, the 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of leukocyte DNA was measured by high-performance liquid chromatography electrochemical detection method in 35 age- and sex-matched healthy subjects, 22 undialyzed patients with advanced renal failure, and 109 HD patients to assess the relation between oxidative DNA damage and complement-activating membranes, blood antioxidants, and iron status. Dialysis membranes were classified into complement-activating (cellulose; n = 55) and non-complement-activating (polymethylmethacrylate [PMMA]; n = 35; polysulfone [PS]; n = 19) membranes. We found increased oxidative stress in undialyzed and HD patients based on a decrease in plasma levels of ascorbate and alpha-tocopherol adjusted for blood lipid (alpha-tocopherol/lipid), serum albumin, and reduced glutathione levels in whole blood and an increase in oxidized glutathione levels in whole blood compared with controls (P < 0.001). The greatest 8-OHdG level in leukocyte DNA was in HD patients, followed by undialyzed patients and healthy controls (P < 0.001), and was significantly greater in HD patients using cellulose membranes than those using PMMA or PS membranes (P < 0.001). 8-OHdG levels correlated with plasma alpha-tocopherol/lipid (r = -0.314; P < 0.005), serum iron (r = 0. 446; P < 0.001), and transferrin saturation values (r = 0.202; P < 0.05) in the analysis of all HD patients. In a 6-week crossover study, 8-OHdG levels significantly decreased after the switch from cellulose to synthetic membranes for 2 weeks and increased after the shift from synthetic to cellulose membranes (P < 0.05). Iron metabolism indices and plasma alpha-tocopherol/lipid values did not change significantly in the study period. We conclude that 8-OHdG content in leukocyte DNA is a biomarker of oxidant-induced DNA damage in HD patients. Oxidative DNA damage is a consequence of uremia, further augmented by complement-activating membranes.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Lipophilic antioxidants and iron status in ESRD patients on hemodialysis.

Cardiovascular disease remains the major cause of mortality in hemodialysis patients. Abnormal oxidative stress and impaired antioxidant defense may contribute to accelerated atherogenesis associated with uremia. As oxidative modification of lipids appears to be a prerequisite for the development of atherosclerotic lesions, lipophilic antioxidants may be protective. The aim of this study was to determine the plasma levels of lipophilic antioxidants in 82 hemodialysis patients and 30 controls and to investigate the influence of body iron status on the levels of lipophilic antioxidants. The patients were categorized into 3 groups according to their serum ferritin levels. We found that the plasma levels of lycopene, delta-tocopherol, gamma-tocopherol and retinol of hemodialysis patients were lower than those of controls. On the other hand, both absolute and lipid-normalized plasma lycopene levels were significantly reduced in those patients in the groups with higher ferritin levels as compared to those with lower ferritin levels. In addition, our study showed that the lipid-normalized plasma levels of beta-carotene and alpha-carotene of hemodialysis patients with higher ferritin levels were lower than those of the patients with lower levels. These data suggest that the plasma levels of lipophilic antioxidants are altered in end-stage renal disease on hemodialysis and may be considered as markers of oxidative stress in these patients. Most importantly, elevated serum ferritin levels may affect the levels of these lipophilic antioxidants.

Adult↗
PubMed2000

Morphological and morphometric analysis of human detrusor mitochondria with urodynamic correlation after partial bladder outlet obstruction.

PURPOSE: We correlated ultrastructural changes in mitochondria in the human detrusor with the severity of partial bladder outlet obstruction on urodynamics. MATERIALS AND METHODS: We recruited into the study 52 men with and without bladder outlet obstruction symptoms. The severity of partial bladder outlet obstruction was determined by pressure flow study. Random detrusor biopsy specimens obtained by cystoscopy were fixed immediately and processed for transmission electron microscopic observation. Random areas were photographed for further morphological and morphometric analysis using mitochondrial damage score and stereological principles. RESULTS: Mitochondrial damage score and mean mitochondrial volume strongly correlated with the urodynamic severity of partial bladder outlet obstruction, while mitochondrial volume density, surface density of the mitochondrial outer membrane and number of mitochondria per unit of cytoplasm area did not significantly correlate with severity. CONCLUSIONS: Detrusor mitochondrial swelling and structural destruction increased with the severity of partial bladder outlet obstruction. These changes may be associated with impaired mitochondrial function and oxidative metabolism after partial bladder outlet obstruction. Detrusor mitochondrial damage may explain voiding dysfunction after partial bladder outlet obstruction develops.

Adult↗
PubMed2000

Phenotypic heterogeneity in a Chinese family with mitochondrial disease and A3243G mutation of mitochondrial DNA.

The A3243G mutation of mitochondrial DNA (mtDNA) has been shown to be responsible for or associated with mitochondrial myopathy, encephalopathy, lactic acidosis, strokelike episodes (MELAS) syndrome, diabetes mellitus (DM) and several other neuromuscular diseases. We used polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) to identify the A3243G mtDNA mutation and an electron microscope to examine mitochondrial derangement in the muscle biopsies of a 38-year-old man suspected to have MELAS syndrome with DM. We found great variability in the clinical presentation and in the proportion of mtDNA with the A3243G mutation in the matrilineal family members of the patient. The proband had atypical MELAS syndrome, recurrent vascular headache, and DM (MELASDM), and his mother manifested chronic progressive ptosis and DM (CPPDM). Brain magnetic resonance imaging of the proband showed high signal intensity in the left temporoparieto-occipital area on T2 weighted images (T2WI). The blood lactate level ranged from 2.32 to 4.70 mmol/l, and two-hour postprandial glucose ranged from 124 mg/dl to 148 mg/dl. The blood lactate and postprandial glucose of the proband's mother were 3.15 mmol/l and 192 mg/dl, respectively. Electron microscopic examination of a muscle biopsy of the patient showed abnormal mitochondria with decreased density of cristae and membrane degeneration. No ragged-red fibers were detected in muscle upon staining with modified Gomori trichrome. The hair follicles and blood cells of the patient and his mother showed the A3243G mutation in the tRNA(Leu)(UUR) gene. The proportions of the mutant DNA in the hair follicles and blood cells of the proband were 36.8% and 35.2%, respectively, and those of the patient's mother were 28.8% and 13.9%, respectively. We conclude that the A3243G mtDNA mutation may manifest with MELASDM or CPPDM in different matrilineal members of the same family as a result of differences in random segregation of the heteroplasmic A3243G mutant mtDNA in the affected tissues of patients.

Adult↗
PubMed2000

Absence of maternal A3243G mtDNA mutation and reversible hyperglycemia in a patient with MELAS syndrome.

We report the unusual features of a female patient who had MELAS-specific A3243G mutation in mitochondrial DNA (mtDNA) and diabetes mellitus (DM). The patient showed mitochondrial myopathy, encephalopathy, lactic acidosis, and deafness but lacked the stroke-like episode. Acute hyperglycemia was noted after one attack of status epilepticus. Molecular genetic analysis demonstrated a heteroplasmic A3243G point mutation in the mtDNAs of muscle, blood cells and hair follicles. Glucagon stimulation test exhibited marked depression of pancreatic beta-cell function. However, in a further study neither this mutation, nor MELAS syndrome or DM, was found in all of her maternal relatives. A series of follow-up studies for beta-cell function also showed gradual improvement. The pedigree study led us to believe that this A3243G mutation arose from the germ line cells or occurred later in somatic tissues of the patient. We also suggest that the A3243G mutation of mtDNA may elicit the pathogenesis of a subtype of DM. Nevertheless, environmental stress may be another important factor for provocation of the disease.

Adult↗
PubMed2000

False positive molecular diagnosis of Leber's hereditary optic neuropathy.

BACKGROUND: The most common pathogenic mitochondrial DNA (mtDNA) mutation associated with Leber's hereditary optic neuropathy (LHON) is at the 11,778 nucleotide (nt) position and is usually detected by loss of an Sfa NI restriction site. However, Sfa NI restriction site includes five nucleotides. Substitution of any of the five nucleotides leads to loss of the cutting site and causes a false-positive result. We investigated the false-positive diagnosis of LHON by loss of the Sfa NI restriction site using Sfa NI restriction site analysis and single-strand conformation polymorphism (SSCP) analysis. METHODS: Mae III restriction analysis for double confirmation of the Sfa NI restriction site and direct sequencing for final confirmation of SSCP analysis were performed. RESULTS: The sensitivity of Sfa NI test was 100% and the specificity of the Sfa NI test was 97%. The false-positive rate of Sfa NI test was 3%. SSCP analysis showed 100% sensitivity. Direct sequencing showed 32 patients had a mutation at nt 11,778 of mtDNA and one patient had a silent mutation at nt 11,782 of mtDNA. CONCLUSIONS: These results suggest that restriction enzyme digestion analysis requires double confirmation to avoid a false-positive diagnosis and that DNA sequencing is needed for the confirmation of the mutation detected by SSCP.

Adolescent↗
PubMed1999

Concurrent increase of oxidative DNA damage and lipid peroxidation together with mitochondrial DNA mutation in human lung tissues during aging--smoking enhances oxidative stress on the aged tissues.

Although mutation of mitochondrial DNA (mtDNA) in human tissues has been established to associate with intrinsic aging, the impact of environmental factors on the formation and accumulation of mtDNA mutations and oxidative DNA damage in human tissues is poorly understood. We have investigated the levels of mtDNA with the 4977-bp deletion and A3243G point mutation, oxidative DNA damage (indicated by the formation of 8-hydroxy-2'-deoxyguanosine, 8-OH-dG), and lipid peroxides in lung tissues from smokers and nonsmokers of subjects of different ages. The results showed concurrent age-dependent increase of the 4977-bp deleted mtDNA (P < 0.001), 8-OH-dG (P < 0.05), and lipid peroxides (P < 0.05) in the human lung. In the group of subjects above 60 years old, smokers had more extensive DNA damage and lipid peroxidation than did the nonsmokers. However, the levels of mtDNA with the 4977-bp deletion and A3243G point mutation in the lung of smokers were not significantly different from those of the age-matched nonsmokers. Taken together, these results suggest that accumulation of mtDNA with the 4977-bp deletion together with oxidative DNA damage and lipid peroxides is associated with aging and that smoking enhances oxidative damage in human lung tissues.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin.

Mitochondrial DNA (mtDNA) mutations and impaired respiratory function have been demonstrated in various tissues of aged individuals. We hypothesized that age-dependent increase of ROS and free radicals production in mitochondria is associated with the accumulation of large-scale mtDNA deletions. In this study, we first confirmed that the proportion of mtDNA with the 4977 bp deletion in human skin tissues increases with age. We then investigated the 8-hydroxy-2'-deoxyguanosine (8-OH-dG) content in skin tissues and lipid peroxides content of the skin fibroblasts from subjects of different ages. The results showed an age-dependent increase of 8-OH-dG level in the total DNA of skin tissues of the subjects above the age of 60 years. The specific content of malondialdehyde, an end product of lipid peroxidation, was also found to increase with age. On the other hand, we examined the enzyme activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPx) in the skin fibroblasts. The activities of Cu,Zn-SOD, catalase and glutathione peroxidase were found to decrease with age. However, the activity of Mn-SOD was increased with age before 60 years but was decreased thereafter. Moreover, the activity ratios of Mn-SOD/catalase and Mn-SOD/GPx exhibited the same pattern of change with age. This indicates that free radical scavenging enzymes can effectively dispose of ROS and free radicals before 60 years of age. However, elevated oxidative stress caused by an imbalance between the production and removal of ROS and free radicals occurred in skin fibroblasts after 60 years of age. Taken together, we suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Age-associated alteration of blood thiol-group-related antioxidants in smokers.

Total blood glutathione and nonglutathione free sulfhydryl compounds, glutathione peroxidase, and glutathione S-transferase in erythrocytes and plasma and also lipid peroxides in plasma were investigated in 48 male smokers and 42 male nonsmokers. We found that the level of total blood glutathione was significantly increased in young smokers (age <40) but no such change was noted in aging smokers (age>/=40). The activities of glutathione peroxidase and glutathione S-transferase in plasma were significantly decreased in young smokers and the plasma levels of these two enzymes and nonglutathione free sulfhydryl compounds were more drastically decreased in aging smokers. The average concentration of plasma lipid peroxides of the aging smokers (2.76+/-0.46 nmol/ml) was significantly higher than that of the aging nonsmokers (2.32+/-0. 41nmol/ml, P=0.049). On the other hand, the level of total blood glutathione was negatively correlated with the level of plasma lipid peroxides (r=-0.305, P=0.002) and was positively correlated with the smoking index (r=0.307, P=0.019) of all the study subjects under age control. These results indicate that the activities of glutathione peroxidase and glutathione S-transferase declined to a great extent under smoking-mediated oxidative stress in the blood of both young and aging smokers. Moreover, the compensatory generation of total blood glutathione may effectively prevent plasma lipids from peroxidation in young smokers, although the activities of glutathione peroxidase and glutathione S-transferase in plasma were decreased. By contrast, total blood glutathione was inadequate for such protection in the aging smokers. We suggest that supplementation of thiol-group-related agents may be considered for the prevention or alleviation of oxidative stress in aging smokers, whose capability and capacity for the disposal of smoking-mediated free radicals and reactive oxygen species are compromised.

Adult↗
PubMed1999

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations.

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.

Adenosine Diphosphate↗
PubMed1999

Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.

Adenosine Triphosphatases↗
PubMed1999

Intravenous ascorbic acid as an adjuvant therapy for recombinant erythropoietin in hemodialysis patients with hyperferritinemia.

BACKGROUND: Inadequate iron mobilization and defective iron utilization may cause recombinant erythropoietin (rEPO) hyporesponsiveness in hemodialysis (HD) patients with iron overload. We have demonstrated that intravenous ascorbic acid (IVAA), but not intravenous iron medication, can effectively circumvent the functional iron-deficient erythropoiesis associated with iron overload in HD patients. However, it is uncertain whether all HD patients with hyperferritinemia will consistently respond to IVAA and which index may indicate functional iron deficiency in the special entity. Therefore, a prospective study was conducted to establish the guidelines for IVAA adjuvant therapy. METHODS: Sixty-five HD patients with serum ferritin levels of more than 500 microgram/liter were recruited and divided into the control (N = 19) and IVAA (N = 46) groups. IVAA patients with a hematocrit (Hct) of less than 30% received 300 mg of ascorbic acid three times per week for eight weeks. Controls had a Hct of more than 30% and did not receive the adjuvant therapy. Red blood cell and reticulocyte counts, iron metabolism indices, erythrocyte zinc protoporphyrin (E-ZPP), and the concentrations of plasma ascorbate and oxalate were examined before and following the therapy. RESULTS: Thirteen patients (four controls and nine IVAA patients) withdrew by the end of the study. Eighteen patients had a dramatic response to IVAA with a significant increase in their hemoglobin and reticulocyte index and a concomitant 24% reduction in rEPO dose after eight weeks. This paralleled a significant rise in serum iron and transferrin saturation (TS) and a fall in E-ZPP and serum ferritin (baselines vs. 8 weeks, serum iron 68 +/- 37 vs. 124 +/- 64 microgram/dl, TS 27 +/- 10 vs. 48 +/- 19%, E-ZPP 123 +/- 44 vs. 70 +/- 13 micromol/mol heme, and serum ferritin 816 +/- 435 vs. 587 +/- 323 microgram/liter, P < 0. 05). Compared with responders, mean values of hemoglobin, rEPO dose, iron metabolism parameters, and E-ZPP showed no significant changes in controls (N = 15) and in non-responders (N = 19). Thirty-seven patients (18 responders and 19 non-responders) were further analyzed by receiver operating characteristic curves to seek the criteria for prediction of a response to IVAA treatment. The results showed that E-ZPP at a cut-off level of more than 105 micromol/mol heme and TS at a level of less than 25% were more specific to confirm the status of functional iron deficiency in iron-overloaded patients. The two criterion values had the highest accuracy to predict a response to treatment. CONCLUSIONS: Functional iron-deficient erythropoiesis plays a role in rEPO-hyporesponsive anemia in HD patients with hyperferritinemia. IVAA may be an adjuvant therapy for rEPO in these patients, and E-ZPP of more than 105 micromol/mol heme and TS of less than 25% should be used to guide the IVAA treatment.

Adult↗