Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome

Summary We have compared respiratory chain enzyme activities, ATP synthesis, and ATP hydrolysis in cultured fibroblast mitochondria from patients with Leigh syndrome (LS) due to: (i) cytochrome oxidase (COX) deficiency (#6); (ii) pyruvate dehydrogenase complex (PDHC) deficiency (#4); and (iii) maternally inherited LS (MILS) with the T8993G mutation in the ATPase 6 gene of mtDNA (#5). Enzyme activities were normal in patients with MILS and variably decreased in those with COX and PDHC deficiency. ATP hydrolysis was normal or mildly decreased in all three groups. In contrast, ATP synthesis was decreased in all patients but more markedly in those with MILS, and especially with pyruvate/malate as substrate. These studies show that impaired ATP production is the common feature of all three forms of LS, but it is both more severe and more specific in MILS, consistent with the genetic defect.     

骨髓增生异常综合征线粒体呼吸链酶复合体的变化

目的:研究骨髓增生异常综合征(MDS)患者骨髓单个核细胞线粒体呼吸链的功能变化并分析其与MDS的关系.方法:测定26例MDS患者与10例骨髓象正常者的单个核细胞线粒体呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ的活性.结果:MDS患者呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ的活性明显低于对照组(P＜0.05);线粒体呼吸链酶复合体Ⅰ、Ⅲ的活性在RAEB-Ⅰ、RAEB-Ⅱ组与RA、RARS组的差异无统计学意义(均P＞0.05),而酶复合体Ⅳ的活性在RAEB-Ⅰ、RAEB-Ⅱ组较RA、RARS组高,差异有统计学意义(P＜0.05).结论:线粒体呼吸链酶复合体的活性在MDS患者中降低,可能与MDS的病态造血及无效造血有关.     

The pleiotropic effects of phosphodiesterase 5 inhibitors on function and safety in patients with cardiovascular disease and hypertension

J Clin Hypertens (Greenwich). 2012; 14:644–649. © 2012 Wiley Periodicals, Inc. Phosphodiesterase 5 (PDE‐5) inhibitors are selective blockers of PDE‐5, which catalyzes the hydrolysis of cyclic guanosine monophosphate (cGMP) to its corresponding monophosphates. cGMP is a potent vasodilator and nitric oxide donor. Since PDE‐5 is widely distributed in the body, it was hypothesized that inhibition of its actions could lead to significant vasodilation, which could benefit patients with coronary artery disease. This hypothesis led to the development of PDE‐5 inhibitors, the first being sildenafil citrate. Studies of sildenafil in patients with coronary artery disease demonstrated a modest cardiovascular effect but a potent action on penile erection in men, resulting in sildenafil becoming first‐line treatment of erectile dysfunction. Two more PDE‐5 inhibitors are now US Food and Drug Administration–approved (vardenafil and tadalafil) for the treatment of erectile dysfunction. Recent studies have demonstrated several beneficial pleiotropic cardiovascular effects of PDE‐5 inhibitors in patients with erectile dysfunction and multiple comorbidities, including coronary artery disease, heart failure, hypertension, and diabetes mellitus. Treatment of these conditions with PDE‐5 inhibitors has been very effective, safe, and well tolerated. Drug interactions have been minimal with the exception of nitrates, where coadministration may result in severe vasodilation and hypotension. These beneficial pleiotropic and safe cardiovascular effects of PDE‐5 inhibitors will be discussed in this concise review.     

Acylcarnitine profiles in fibroblasts from patients with respiratory chain defects can resemble those from patients with mitochondrial fatty acid beta-oxidation disorders

Mitochondrial fatty acid beta-oxidation (FAO) is coupled to the respiratory chain (RC). Functional defects of one pathway may lead to secondary alteration in flux through the other. We investigated the acylcarnitine profiles in cultured fibroblasts obtained from 14 healthy subjects, 31 patients with 8 different primary enzyme deficiencies of FAO, and 16 patients with primary RC defects including both isolated and multiple enzyme complex defects. Intact cells were incubated in media containing deuterium-labeled hexadecanoic acid and L-carnitine, and the acylcarnitines analysed using an electrospray tandem mass spectrometer. All FAO-deficient cell lines revealed disease-specific acylcarnitine profiles related to the sites of defects. Some cell lines from patients with RC defects showed profiles similar to those of controls, whereas others had abnormal profiles mimicking those found in FAO disorders. The acylcarnitine profiles of patients with RC enzyme defects were not predictable, and in some patients defects caused by mutations in either nuclear-encoded or mitochondrial DNA were associated with acylcarnitine abnormalities. While in vitro acylcarnitine profiling is useful for the diagnosis of FAO deficiencies, abnormal profiles do not exclusively indicate these disorders, and primary defects of the RC remain a possibility. Awareness of this diagnostic pitfall will aid in the selection of subsequent confirmatory tests and therapeutic options.     

NormalN-oligosaccharyltransferase activity in fibroblasts from patients with carbohydrate-deficient glycoprotein syndrome

Summary TheN-oligosaccharyltransferase activity was found to be normal in fibroblasts from five patients with the carbohydrate-deficient glycoprotein syndrome type I and type III using an assay based on dolichyldiphosphoryl-N-N-diacetylchitobiose as donor and a hexapeptide as acceptor substrate.     

Decreased deoxyribonucleic acid binding of glucocorticoid-receptor complex in cultured skin fibroblasts from a patient with the glucocorticoid resistance syndrome

A patient with the syndrome of glucocorticoid resistance was studied. A 27-yr-old woman initially was diagnosed as having Cushing's disease, based on the findings of high plasma ACTH and serum cortisol levels, increased urinary cortisol secretion, resistance to adrenal suppression with dexamethasone, and bilateral adrenal hyperplasia by computed tomography and scintigraphy of the adrenal glands. However, she had no signs or symptoms of Cushing's syndrome. During a 5-yr follow-up, no clinical abnormalities developed, although hypercortisolism persisted. End-organ resistance to cortisol was suspected. To explain the end-organ resistance to cortisol, the glucocorticoid receptors (GR) in peripheral mononuclear leukocytes and cultured skin fibroblasts from a forearm skin biopsy were characterized and compared with the results of similar studies in normal subjects. The patient's GR in whole cell assays had an increased dissociation constant (Kd). In the cytosol of cultured skin fibroblasts from the patient, there was also decreased binding capacity. The thermal stability and the sedimentation coefficient in a sucrose density gradient of the receptors in the cytosol of cultured skin fibroblasts from the patient and normal subjects were similar. GR complex activation, analyzed by DEAE-cellulose chromatography, was decreased in the patient. DNA binding of the GR complex after temperature-induced activation was lower in the patient than in normal subjects. Nuclear translocation of GR complexes from the patient was also slightly decreased. These results suggest that the patient's glucocorticoid resistance was due to a decrease in the affinity of the receptor for glucocorticoids and a decrease in the binding of the GR complex to DNA.     

体外反搏对代谢综合征患者血管内皮功能的影响

目的探讨体外反搏对代谢综合征(MS)患者血管内皮功能及胰岛素抵抗的影响及其作用机制。方法MS患者50例随机分为两组,增强型体外反搏(EECP)组20例,对照组30例。两组均进行健康教育,血压不低于150/95mmHg的患者给予贝那普利治疗,在此基础上,EECP组给予1h/d,共36h的体外反搏治疗。两组治疗前后测定血压、空腹血糖、血脂、胰岛素、胰岛素敏感指数(ISI)及肱动脉血管内皮依赖性舒张功能。结果两组治疗后内皮依赖性舒张功能均有改善(P<0.01),但EECP组改善更明显(Z=-3.743,P<0.01);EECP组患者治疗后空腹血糖、血清胰岛素水平明显下降(P<0.01),ISI明显升高(P<0.01),血脂代谢紊乱改善,甘油三酯降低(P<0.01),高密度脂蛋白胆固醇升高(P<0.05)。结论体外反搏通过提高血流切应力,可明显改善MS患者血管内皮依赖性舒张功能,改善胰岛素抵抗,为体外反搏的临床应用提供进一步的理论依据。     

Lipid lowering versus pleiotropic effects of statins on skin microvascular function in patients with dysglycaemia and coronary artery disease

Objectives. To investigate the impact of lipid lowering therapy by different means on skin microvascular function in patients with dysglycaemia and coronary artery disease (CAD). Design and setting. Thirty‐six patients were randomized to simvastatin 80 mg daily (S80, n = 19) or ezetimibe 10 mg and simvastatin 10 mg daily (E10/S10, n = 17) for 6 weeks. Skin microvascular function was assessed by laser Doppler fluxmetry (LDF) at rest, following arterial occlusion (peak postocclusive LDF) and following local heating on the forearm (heat arm LDF) and foot (heat foot LDF). LDF parameters and serum lipids were evaluated at baseline and follow‐up. Results. At follow‐up, LDL cholesterol had decreased from 3.1 (2.7–3.5) to 1.6 (1.5–1.8) (mmol L^?1) and 3.0 (2.4–3.9) to 1.3 (1.1–1.8) (mmol L^?1) in the E10/S10 and S80 groups respectively. In the entire study group (n = 32), LDF parameters increased significantly; postocclusive LDF from 22 (17–27) to 26 (21–32) perfusion units (PU) (P < 0.001), heat foot LDF from 61 (44–82) to 66 (45–83) PU (P < 0.001) and heat arm LDF from 60 (48–121) to 75 (54–125) PU (P < 0.01). The changes in LDF parameters did not differ between the E10/S10 and S80 groups. Conclusions. Lipid lowering improves microvascular function in patients with dysglycaemia and CAD. The data suggest that lipid lowering per se is more important than pleiotropic effects of statins for this effect.     

13-cis-Retinoic acid treatment in patients with myelodysplastic syndrome

Twenty patients with primary myelodysplastic syndromes (16 refractory anemia without or with ringed sideroblasts, 2 refractory anemia with excess blasts, 2 refractory anemia with excess blasts in transformation) received 13-cis-retinoic acid at a dosage of 50-100 mg/m2/day for a minimum of 4 weeks. Twelve patients obtained an increase of hemoglobin levels greater than 1 g/dl and 7 showed an associated increase of granulocyte count greater than 50% of baseline values. No significant biochemical signs of dyslipidemia or liver damage were noted. A sustained response was noted only in refractory anemia without or with ringed sideroblasts and normal or hypercellular bone marrow. Five patients are still on therapy from 23 to 82 weeks without transfusion requirement and all have shown an improvement in performance status. We conclude that 13-retinoic acid may only be clinically useful in selected patients since in myelodysplastic syndromes with blast excess the drug does not seem to improve the course of the disease.     

Decreased procollagen production in cultured fibroblasts from patients with Lowe's syndrome

The oculo-cerebro-renal syndrome described by Lowe is a catastrophic disease in children characterized by progressive eye, central nervous system and kidney degeneration. We determined procollagen production in cultured skin fibroblasts originating from patients with Lowe's syndrome as well as normal individuals after incubation of cells with [¹⁴C]proline for 1, 4 and 20 h. Using [¹⁴C]hydroxyproline formation, in relation to cell protein or DNA, as an index of procollagen production, we found that cultured cells from patients synthesized collagenous protein at a substantially reduced level. This decrease in synthesis in cultured fibroblasts from patients could not be accounted for by differences in the specific radioactivity of the free intracellular proline; moreover, it could not be attributed to differences in the kinetics of growth between normal and affected cell lines. The degree of hydroxylation of proline residues in collagen was the same in both normal and affected cells.     

Altered fibronectin distribution in cultured fibroblasts from patients with Ehlers-Danlos syndrome

Using the immunofluorescence technique we have studied the distribution of fibronectin in cultured fibroblasts from patients affected by Ehlers-Danlos Syndrome (EDS) types I, II and VI. In these cells the amount of fibronectin production is reduced with respect to normal fibroblasts; moreover fibronectin fibers are shorter, thicker, mainly pericellular and show intracytoplasmic accumulation. The altered fibronectin distribution may be the result of altered interactions of fibronectin with extracellular matrix components, either due to abnormal fibronectin or to changes in other extracellular matrix molecules (e.g. collagens, hyaluronic acid).     

The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders

Summary The oxidation of pristanic and phytanic acids by human skin fibroblasts was compared to that of their synthetic analogues, 2-methylpalmitic and 3-methylmargaric acids. The synthetic compounds and natural substrates were degraded at comparable rates in control and X-linked adrenoleukodystrophy fibroblasts. The -decarboxylation of 3-methylmargaric acid, similarly to that of phytanic acid, was affected in Refsum disease and Zellweger syndrome, but not in X-linked adrenoleukodystrophy. The -oxidation of 2-methylpalmitic acid, similarly to that of pristanic acid, was deficient in fibroblasts derived from patients suffering from Zellweger syndrome, confirming the importance of peroxisomes in the breakdown of 2-methyl-branched fatty acids. No deficiency was observed in fibroblasts from X-linked adrenoleukodystrophy patients. The 1-¹⁴C-labelled 2- and 3-methyl-branched fatty acids, which are easier to synthesize that the natural analogues, are therefore valuable tools for the diagnosis of human peroxisomal disorders.     

Lipid-altering changes and pleiotropic effects of atorvastatin in patients with hypercholesterolemia

In this prospective study, we found beneficial short-term effects from atorvastatin therapy, including effects on low-density lipoprotein subfractions and remnant-like lipoprotein particle cholesterol, antioxidant effects, and alterations in endothelial function that may be important in early benefit from statin therapy; some effects would support much earlier benefit than previously reported. We also found long-term effects of atorvastatin, including decreased plasminogen activator inhibitor type-1 and additional significant alterations in low-density lipoprotein subfractions and endothelial function, supporting benefits from continuous long-term atorvastatin therapy beyond early reversal of hypercholesterolemia.     

Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I

A comparison of the clinical presentation, disease course and results of laboratory and imaging studies of all patients so far published with a NDUFS4 mutation are presented. This reveals marked clinical heterogeneity, even in patients with the same genotype.     

普罗布考对急性冠状动脉综合征患者血管内皮损伤的干预效应

目的 观察普罗布考早期治疗对急性冠状动脉综合征（ACS）患者的血管内皮功能的影响。方法 49例ACS患者随机分为两组,普罗布考组（P组,n=24）和常规治疗组（C组,n=25）,检测外周血氧化低密度脂蛋白（ox-LDL）、一氧化氮（NO）及循环内皮细胞（CEC）数量,应用高频超声测肱动脉内皮依赖血管舒张功能（FMD）,用药治疗3个月,观察治疗前后指标变化。结果 P组患者治疗后与治疗前相比,外周血中ox-LDL和CEC水平明显降低（P〈0．01）,NO含量明显升高（P〈0．01）,FMD明显升高（P〈0．05）。P组患者治疗后与C组相比,外周血中ox-LDL和CEC水平明显降低（P〈0．01,P〈0．05）,NO明显升高（P〈0．01）,FMD明显升高（P〈0．05）。C组CEC、NO含量及FMD含量治疗前后差异无统计学意义（P〉0．05）。P组治疗后血浆ox-LDL与CEC呈正相关（r=0．385,P〈0．01）,与NOFMD呈负相关（r=-0．517,P〈0．01）。结论 普罗布考具有抗氧化、降低ox-LDL,提高ACS患者血管内皮功能的作用。     

Assessment of endothelial function in complex regional pain syndrome type I

The objective of this study is to investigate the endothelial function in complex regional pain syndrome. A total of 21 patients with CRPS and 15 healthy controls were enrolled. The mean age of patients was 22 ± 4.9 years and the mean duration of the disease was 5.9 ± 2.5 months. Flow-mediated vasodilatation (FMD) technique was performed for evaluating the endothelial function. Parameters were the waveforms obtained and the increase in diameter and blood flow of the brachial artery, which were expressed as the percent change from baseline. The differences of the waveforms obtained in the affected limbs in comparison to non-affected limbs and to controls were statistically significant. We observed a trend of greater percent dilating responses in the affected limbs; however, the differences showed no statistical significance. In macrovascular evaluation, the endothelial function seems to be impaired in the earlier stage of the disease.