Oxidative damage to mitochondrial DNA and its relationship to diabetic complications

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Oxidative stress is known to increase the conversion of deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OHdG) in DNA, which is linked to increased mitochondrial DNA (mtDNA) deletions. We investigated mtDNA deletions and 8-OHdG in the muscle DNA of non-insulin-dependent diabetes mellitus (NIDDM) patients. mtDNA deletion of 4977 bp (delta mtDNA4977) and the content of 8-OHdG in the muscle DNA of the NIDDM patients were much higher than those of the control subjects. There was a significant correlation between delta mtDNA4977 and the 8-OHdG content (P < 0.0001). Both delta mtDNA4977 and the 8-OHdG content were also correlated with the duration of diabetes. Delta mtDNA4977 and the 8-OHdG content in muscle DNA increased in proportion to the severity of diabetic nephropathy and retinopathy. This is the first report that an increase in delta mtDNA4977 and 8-OHdG is proportional to the severity of diabetic complications. Oxidative mtDNA damage is speculated to contribute to the pathogenesis of diabetic complications though a defect in mitochondrial oxidative phosphorylation or other mechanisms. 8-OHdG and delta mtDNA4977 are useful markers to evaluate oxidative mtDNA damage in the diabetic patients.     

Multiple skeletal muscle mitochondrial DNA deletions in patients with unilateral peripheral arterial disease

Peripheral arterial disease (PAD) is associated with metabolic derangements and accumulation of the common 4977 bp mitochondrial DNA (mtDNA) deletion mutation. The current study was undertaken to test the hypothesis that PAD is associated with multiple mtDNA deletions. Gastrocnemius biopsies were obtained from nine patients with unilateral PAD. DNA extracted from the biopsies was analyzed for mtDNA deletions using a primer-shift PCR strategy. Multiple primers and strict, prospective criteria were used to identify deletions. PAD was associated with multiple mtDNA deletions (average of 8.2 distinct deletions in muscle from the hemodynamically affected limb). mtDNA injury was present in both the worse- and less-affected limbs of the unilateral PAD patients, and the estimated degree of mtDNA injury was strongly correlated in the two limbs on an intra-subject basis. The 4977 bp deletion was frequently identified, but was not always the deletion of highest frequency in individual samples. The estimated relative frequency of the 4977 bp deletion was correlated with the overall mtDNA injury in the biopsies. In summary, PAD is associated with mtDNA injury as reflected by multiple deletion mutations. As the mutations are not limited to the ischemic limb in unilateral patients, they are unlikely to contribute to the pathophysiology of claudication.     

病毒性心肌炎与扩张型心肌病心肌线粒体DNA缺失的定量分析

目的对比研究病毒性心肌炎(VMC)与扩张型心肌病(DCM)患者活检心肌组织线粒体DNA(mtDNA)缺失突变情况及其与外周淋巴细胞mtDNA缺失程度的相关性.方法用定量PCR法检测20例VMC患者、12例DCM患者心肌细胞及其外周血淋巴细胞mtDNA4977碱基对(mtDNA4977)和mtDNA7436碱基对(mtDNA)缺失率.取12例健康意外死亡者心肌和23例献血员外周血淋巴细胞作正常对照.结果正常对照者、VMC和DCM患者心肌细胞均存在mtDNA4977及mtDNA7436缺失,合计缺失率分别为0.175%、0.385%和3.004%;外周淋巴细胞mtDNA缺失程度与心肌细胞呈一致性改变,且有良好的相关性(r=0.960,P＜0.001).结论mtDNA缺失可能是VMC发病及其向DCM演变的一个重要心肌损伤机制;外周淋巴细胞在研究心肌细胞mtDNA缺失中的作用值得进一步探讨.     

病毒性心肌炎患者心肌细胞线粒体DNA缺失的定量分析

为探讨病毒性心肌炎 (VMC)患者心肌细胞线粒体 DNA(mt NDA)缺失突变情况及意义 ,用定量 PCR法检测 2 0例 VMC患者心肌细胞及其外周血淋巴细胞 mt DNA4 977碱基对 (mt DNA4 977)和 mt DNA74 36 碱基对 (mt D-NA74 36 )缺失率。取 10例健康意外死亡者心肌和 2 0例献血员外周血淋巴细胞作正常对照。结果显示 ,正常对照者和 VMC患者心肌细胞均存在 m t DNA4 977及 mt DNA74 36缺失 ,合计缺失率分别为 0 .176 %、0 .384 % ,二者差异显著 ,P<0 .0 5 ;VMC患者外周淋巴细胞 mt DNA缺失程度与心肌细胞呈一致性改变 ,且有良好的相关性 (r=0 .92 0 ,P<0 .0 0 1)。提示 mt DNA缺失可能是 VMC发病过程中重要的心肌损伤机制 ;外周淋巴细胞在研究心肌细胞 mt DNA缺失中的作用值得进一步探讨     

Mitochondrial DNA deletions and the aging heart

Mitochondrial DNA (mtDNA) mutations appear to be associated with a wide spectrum of human disorders and proposed to be a potential contributor of aging. However, in an age-dependent increase of the common 4977 bp deletion of human mtDNA still many unanswered questions remain. Comparing mtDNA copy levels in different tissues revealed that cardiac muscle had the highest, while the cortex cerebelli showed the lowest copy number of mtDNA in every donor. Intriguingly, mtDNA copy number showed no changes during aging. In heart tissue, the amount of 4977 bp mtDNA deletion increased in an age-dependent manner showing significant differences at the age of 40 years and older (p<0.005). In vitro studies analyzing human normal cells transfected with telomerase (BJ-T) revealed that oxidative stress (OS)--a well accepted promoter of aging--induced 4977 bp deletion and point mutations as demonstrated by real-time PCR and DHPLC analysis. Interestingly, OS induced apoptosis only in transformed human fibroblasts by activation of the intrinsic (mitochondrial-mediated) signalling pathway as indicated by morphological damage of mitochondria, DNA laddering and increase of the Bax/Bcl-2 ratio. In conclusion, in heart tissue, the amount of the 4977 bp deletion increased in an age-dependent manner and it was more detectable after the 4th decade of life, although there was some scatter in the data. Since, apoptosis was induced by the mitochondria-mediated pathway only in transformed cells, the role for apoptosis in normal tissue of the aging heart remains unclear.     

Das mitochondriale Genom und Altern

There is a lot of evidence that age-associated alterations of the mitochondrial genome occur, especially in postmitotic tissues such as brain, heart and skeletal muscle. These alterations are supposed to be a result of an attack of free radicals generated as normal byproducts of oxidative phosphorylation and lead to damage of proteins, lipids, and DNA. The alterations of mtDNA include oxidative damage of base pairs, point mutations, large-scale deletions or duplications. The 4977 bp deletion or "common deletion" reveals an age-dependent accumulation in postmitotic tissues, but not in fast-dividing tissues such as blood cells. In addition, it is observed that a tissue-specific accumulation occurs with the highest abundance in the basal ganglia, followed by skeletal muscle, heart, and lowest in cerebellar tissue. Third, pathological alterations of specific tissue, like ischemia/reperfusion events, display a pronounced accumulation of the deletion compared to age-matched controls. Because there are many mtDNA mutations, further analysis of all alterations of mtDNA will elucidate its role in the phenomenon of aging. Despite some criticisms of this free radical theory of aging, there is a lot of experimental evidence to support the important role of mitochondria in organismal aging.     

Oxidative stress,antioxidant status and lipid profile in pulmonary tuberculosis patients before and after anti-tubercular therapy

BackgroundPulmonary tuberculosis (PTB) is a highly infectious dreadful disease caused by mycobacterium tuberculosis (MTB). Numerous studies reported free radicals activity, antioxidant status and lipid profile in PTB patients, but previous studies have lacunae in comparing the biochemical variables between before and after anti-tubercular therapy (ATT) supplementation to PTB patients. Hence, the present study was carried out to investigate oxidative stress markers, antioxidant status, lipid profile, liver function markers, and glycoprotein components in pulmonary tuberculosis patients (PTB) patients before and after 60 days of ATT.MethodsThis is a case-control study carried out with 100 healthy subjects and 110 PTB patients. All the patients diagnosed with sputum test and were positive for acid fast bacilli (AFB) were included for the study. An informed consent was obtained from all the patients.ResultsOur study found increased levels of oxidative stress markers, decreased enzymatic and non-enzymatic antioxidants, altered lipid profile in PTB patients as compared to healthy subjects before treatment and these levels were restored after clinical improvement with ATT. We also found increased concentrations of liver function parameters and components of glycoprotein in PTB patients. ATT refurbished lipid levels, antioxidant status and oxidative stress markers with decrease in liver function enzymes and glycoproteins in PTB patients.ConclusionCo-supplementation of antioxidants, along with ATT and inclusion of nutritious diet could be useful to reduce the pathogenesis of PTB and is warranted as a future study for the management of PTB.     

The frequency of 4977 base pair deletion of mitochondrial DNA in various types of liver disease and in normal liver

Using a polymerase chain reaction (PCR) method, we tested for the hepatic mitochondrial DNA (mtDNA) deletion in 40 hepatic tumors (28 hepatocellular carcinomas [HCcs], 9 other malignant tumors, and 3 benign tumors) and in the livers of 71 patients, including 16 pediatric patients with end-stage liver disease who underwent living related donor liver transplantation and 16 liver donors. A 4977 base pair (bp) deletion of mtDNA was detected in 36 of 55 specimens of non-tumor portions of adult liver (65.5%). However, none of the specimens obtained from cirrhotic livers of the 16 pediatric patients younger than 13 years of age had the 4977 bp deletion. The frequency of mtDNA deletion was significantly decreased compared with normal liver in HCCs (7 of 28) and other malignant liver tumors (2 of 9). The frequency of this deletion was unrelated to the presence of liver cirrhosis, patient's gender, hepatitis B virus surface antigen status, and hepatitis C virus antibody status.     

Detection of the 4977 bp deletion of mitochondrial DNA in different human blood cells

As recently reported, it is possible to detect and quantify the amount of the deleted human mitochondrial DNA (mtDNA) in whole blood, platelets and peripheral blood mononuclear cells using real-time PCR. The aim of this study was to identify the cell types in human blood carrying the 4977 bp deleted mtDNA and their accumulation with regard to donor age. Whole blood from 10 healthy donors (five individuals aged from 19 to 22 years, five aged from 57 to 61 years) was separated in various cell populations such as granulocytes, B cells/monocytes and T cells. Purity of the cell isolates was determined by flow cytometry. Total DNA was extracted and 250 ng DNA of each cell type was subjected to PCR using fluorescent-labelled primer pairs. The specific PCR product of the 4977 bp deletion was quantified using an automated detection system. The accumulation of the 4977 bp deletion was more pronounced in T lymphocytes and granulocytes in comparison to B lymphocytes/monocytes. The amount of the 4977 bp deletion in whole blood varied from 0 to 0.00018%, in T lymphocytes from 0.00009 to 0.00160%, in granulocytes from 0 to 0.00162% and in the B lymphocyte/monocyte fraction from 0 to 0.00025%. The higher amount of the deletion in T lymphocytes may be due to a subset of lymphocytes with a longer lifespan thus facilitating the accumulation of mitochondrial damage. The higher amount in granulocytes could have the explanation in the higher release of free radicals for prevention of infectious diseases, because free radicals are supposed to damage the macromolecules of this cell type. The 10 donors displayed differences in the pattern of the accumulation with regard to the different cell types, but no age-dependent accumulation was observed. Differences of the accumulation pattern may be due to actual individual living behaviour or environmental factors.     

康欣口服液对老年小鼠肾线粒体DNA缺失突变的影响

目的 研究康欣口服液对老年Balb/c小鼠肾线粒体DNA（mtDNA）缺失突变的影响.方法 老年Balb/c小鼠随机分为空白组和康欣口服液组,分别给予生理盐水和康欣口服液4个月.采用聚合酶链反应（PCR）技术和光密度扫描检测两组mtDNA的缺失突变情况.结果 与空白对照组比较,康欣口服液能显著减少老年Balb/c小鼠肾mtDNA的缺失（P＜0.001）.结论 康欣口服液可以抑制老年小鼠mtDNA的缺失突变,对mtDNA有保护作用.     

Effects of oxidative stress on mitochondrial content and integrity of human anastomotic colorectal dehiscence: a preliminary DNA study

BACKGROUND:

Anastomotic dehiscence is one of the most severe complications of colorectal surgery. Gaining insight into the molecular mechanisms responsible for the development of anastomotic dehiscence following colorectal surgery is important for the reduction of postoperative complications.

OBJECTIVE:

Based on the close relationship between surgical stress and oxidative stress, the present study aimed to determine whether a correlation exists between increased levels of reactive oxygen species and colorectal anastomotic dehiscence.

METHODS:

Patients who underwent surgical resection for colorectal cancer were divided into three groups: patients with anastomotic dehiscence (group 1); patients without dehiscence who underwent neoadjuvant radiochemotherapy (group 2); and patients without anastomotic dehiscence who did not undergo neoadjuvant radiochemotherapy (group 3). Quantitative polymerase chain reaction and real-time polymerase chain reaction assays were performed to measure nuclear DNA and mitochondrial DNA (mtDNA) content, and possible oxidative damage to nonmalignant colon and rectal tissues adjacent to the anastomoses.

RESULTS:

mtDNA content was reduced in the colon tissue of patients in groups 1 and 2. Rectal mtDNA was found to be more damaged than colonic mtDNAs in all groups. The 4977 bp common deletion was observed in the mtDNA of tissues from both the colon and rectum of all patients.

DISCUSSION:

Patients in groups 1 and 2 were more similar to one another than to group 3, probably due to higher levels of reactive oxygen species in the mitochondria; the greater damage found in the rectum suggests that dehiscence originates primarily from the rectal area.

CONCLUSIONS:

The present study of mtDNA analyses of normal human colon and rectal tissues from patients with colorectal cancer is among the first of its kind.     

A maternal line study investigating the 4977-bp mitochondrial DNA deletion

The most frequently reported species of mitochondrial DNA (mtDNA) damage associated with ageing is the 4977-bp 'Common Deletion'. However, recent observations have raised several issues within the deletion debate namely: the significance of the 4977-bp deletion (CD) as a universal DNA marker of ageing and mitochondrial dysfunction; and the possibility for maternal transmission of deletions in humans. Previous attempts at answering these questions have been limited because many investigations have been cross-sectional studies of unrelated individuals. With the unique feature of the maternal inheritance of mtDNA, our study overcomes some of these limitations by investigating the CD in human maternal lines, which represent 21 families spanning four generations. Using a highly sensitive PCR methodology, we identified the presence of the CD in leukocytes from all 71 individuals (age range-8 months-99 years) including all infants and children (n=15) which in addition were free of any known mitochondrial diseases. This is important because the few reports of the CD in infants have been linked to mitochondrial disease. These results question the significance of the CD as a universal DNA marker of ageing and subsequent mitochondrial dysfunction and provide support for the possibility for maternal transmission of deletions.     

心肌mtDNA4977缺失与扩张型心肌病猝死的关系

目的探讨扩张型心肌病(DCM)猝死者心肌线粒体DNA(mtDNA)4977缺失情况及其与猝死的关系.方法从近7年尸检案例中挑选11例DCM猝死和14例对照组病例及心肌组织蜡块,按常规方法提取心肌mtDNA,用PCR、琼脂糖紫外凝胶成像技术确定扩增产物激光密度,初步定量检测mtDNA4977缺失率.结果DCM猝死11例中,未成年人2例;成人9例,均为男性,年龄22～49(平均38)岁.对照组14例中,未成年人1例;成人13例,其中男11例,女2例,年龄19～47(平均37.23)岁,死因为机械性损伤和窒息各2例,电击死2例,中毒2例,非心肌病猝死6例.DCM11例(占100%)、对照组2例(占14.28%)检见不同程度的mtDNA4977缺失;两组病例mtDNA4977缺失率均值分别为0.92%和0.09%,差异有统计学意义.结论DCM猝死者心肌可检见mtDNA49 77缺失;其心肌mtDNA4977缺失突变与DCM猝死有关.     

Short-Term Heart Variability and Oxidative Stress in Newly Diagnosed Essential Hypertension

We sought to evaluate oxidative stress parameters like malondialdehyde, total antioxidant status, and time, frequency domain heart rate variability indices in newly diagnosed untreated hypertension. We also sought to study the correlation between heart rate variability and oxidative stress parameters in normotensive and newly diagnosed untreated essential hypertensives. Fourteen normotensive males and 36 newly diagnosed male hypertensives were enrolled for the study. Malondialdehyde, total antioxidant status, and heart rate variability in the frequency and time domain were studied in the hypertensive and normotensive group. Malondialdehyde was significantly higher in the hypertensive group, whereas total antioxidant status was significantly higher in the control group. SDNN, RR triangular index, RMSSD, log ₁₀ low-frequency power, log ₁₀ high-frequency power, log ₁₀ total power, and heart rate variability during deep breathing test was significantly lower in hypertensive patients compared to normotensive controls. SDNN, RMSSD, high-frequency power, and high-frequency power normalized had significant negative correlation with malondialdehyde. Low-frequency power normalized positively correlated with malondialdehyde. Total antioxidant status had a significant negative correlation with blood pressure. The study gives further evidence for a decreased heart rate variability and elevated oxidative stress in hypertension.     

The 4977 bp deletion of mitochondrial DNA in human skeletal muscle,heart and different areas of the brain: A useful biomarker or more?

It has been suggested that deletions of mitochondrial DNA (mtDNA) are important players with regard to the ageing process. Since the early 1990s, the 4977 bp deletion has been studied in various tissues, especially in postmitotic tissues with high energy demand. Unfortunately, some of these studies included less than 10 subjects, so the aim of our study was to quantify reliably the deletion amount in nine different regions of human brain, heart and skeletal muscle in a cohort of 92 individuals. The basal ganglia contain the highest deletion amounts with values up to 2.93% and differences in deletion levels between early adolescence and older ages were up to three orders of magnitude. Values in frontal lobe were on average an order of magnitude lower, but lowest in cerebellar tissue where the amount was on average only 5 x 10(-3) of the basal ganglia. The deletion started to accumulate in iliopsoas muscle early in the fourth decade of life with values between 0.00019% and 0.0035% and was highest in a 102-year-old woman with 0.14%. In comparison to skeletal muscle, the overall abundance in heart muscle of the left ventricle was only one-third. The best linear logarithmic correlation between amount of the deletion and age was found in substantia nigra with r=0.87 (p<0.0005) followed by anterior wall of the left ventricle (r=0.82; p<0.0005). With regard to mitochondrial DNA damage, we propose to use the 4977 bp deletion as an ideal biomarker to discriminate between physiological ageing and accelerated ageing. The biological meaning of mitochondrial deletions in the process of ageing is under discussion, but there is experimental evidence that large-scale deletions impair the oxidative phosphorylation in single cells and sensitize these cells to undergo apoptosis.     

抗衰1号对老年小鼠肝线粒体DNA缺失的影响

目的研究抗衰1号对老年Balb/c小鼠肝线粒体DNA(mtDNA)缺失突变的影响.方法老年Balb/c小鼠随机分为老年空白组和抗衰1号组,分别给予生理盐水和抗衰1号4个月.采用聚合酶链反应(PCR)技术和光密度扫描检测两组mtDNA的缺失情况.结果与老年空白对照组比较,抗衰1号能显著减少老年Balb/c小鼠mtDNA的缺失(P＜0.001). 结论抗衰1号可以抑制老年小鼠mtDNA的缺失,提示补肾健脾活血通腑法组方能减少mtDNA的氧化损伤,对mtDNA有保护作用,从而从分子水平提供了本法延缓衰老的可能机理.     

Atrial fibrillation is associated with accumulation of aging-related common type mitochondrial DNA deletion mutation in human atrial tissue

STUDY OBJECTIVE: Accumulation of somatic mutations of mitochondrial DNA (mtDNA) contributes to the aging process and progressive organ dysfunction. We investigated the mitochondrial DNA with 4977-base-pair mtDNA deletion mutation (mtDNA(4977)) in human atrial tissue and correlated the amount of mtDNA(4977) to clinical atrial fibrillation (AF). METHODS AND RESULTS: Atrial tissue from the right atrial appendage was obtained in 88 patients during open-heart surgery (22 children/adolescents and 66 adults). The amount of mtDNA(4977) was measured using a nested polymerase chain reaction protocol and normalized to wild-type mtDNA. We found that the mtDNA(4977) was absent in all 22 pediatric/adolescent patients. In the adult group, the relative amount of mtDNA(4977) was significantly higher in patients with AF than in patients without AF (0.55 +/- 0.26 vs 0.35 +/- 0.29, p < 0.007) [mean +/- SD]. The amount of mtDNA(4977) was also positively associated with age (r = 0.29, p < 0.01). Left and right atrial pressures, left atrial dimension, hypertension, and cardiac diagnosis did not influence the amount of mtDNA(4977) significantly. Further multivariate analysis showed that both aging and AF contributed independently to the accumulation of mtDNA(4977). CONCLUSION: AF is associated with an increase of mtDNA(4977). This change is similar to the aging process of atrial tissue and might contribute to atrial dysfunction in AF.     

Mitochondrial DNA oxidation and manganese superoxide dismutase activity in peripheral blood mononuclear cells from type 2 diabetic patients

AIM: To investigate the balance between parameters of oxidative stress and antioxidant defences in the mitochondria of peripheral blood mononuclear cells (PBMCs) of type 2 diabetic patients with late complications. METHODS: Ten type 2 diabetic patients with late diabetic complications and 10 age-matched healthy volunteers (controls) were prospectively recruited. Mitochondrial DNA (mtDNA) oxidative damage and mtDNA content were measured as indices of oxidative stress. Manganese superoxide dismutase (MnSOD) activity has been used as an index of mitochondrial antioxidant defence. Mitochondrial respiratory-chain function (cytochrome C oxidase activity) was also assessed. RESULTS: Mitochondrial DNA (mtDNA) oxidation was significantly higher in the PBMCs of diabetic patients than in control subjects (P<0.0001) and, although mtDNA content was lower in the diabetic group, this was not statistically significant. MnSOD activity was significantly increased in PBMCs of type 2 diabetic patients compared with healthy controls (1366+/-187 versus 686+/-167 U/g of protein; P=0.01), and was related to mtDNA oxidative damage. No differences in mitochondrial respiratory-chain function were found between diabetic patients and controls. CONCLUSION: PMBCs from type 2 diabetic patients with late diabetic complications exhibit high mtDNA oxidative damage. The degree of mtDNA oxidation was associated with an increase in MnSOD as an adaptive response to oxidative stress. The consequences of mtDNA oxidative damage on PBMC function and the progression of diabetic complications remain to be elucidated.     

Mitotic brain cells are just as prone to mitochondrial deletions as neurons: a large-scale single-cell PCR study of the human caudate nucleus

Mitochondria are considered a key element in the process of organismic aging, because of their fundamental role in cellular energy generation. In the course of oxidative phosphorylation, harmful free radicals are continuously produced damaging the mitochondrial (mt) genome. One of the consequences is the occurrence of large-scale deletions in mtDNA molecules. The 4977 bp common deletion accumulates exponentially with age, in a mosaic pattern, especially in postmitotic tissues. In order to investigate whether certain cell characteristics underlie this pattern of distribution, and to look for possible age-related changes, two cell types in the caudate nucleus of the human brain from five young and five senescent subjects were analysed by single-cell PCR.

MAP2-positive neurons and GFAP-positive astrocytes were isolated by micromanipulation. For each of the 10 cases, at least 30 cells of each type were collected and subjected to PCR individually. Screening for the presence of the common deletion yielded no significant differences in relative distribution, neither between astrocytes and neurons, nor between healthy young and old humans. Our results imply that the age-dependent increase of the common deletion cannot come about by an increase of independent deletion events in a greater proportion of cells, and that mitotic rate is not a major cellular risk factor for deletion accumulation in the caudate nucleus.     

A novel mtDNA deletion in an infant with Pearson syndrome

Summary Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.