A novel splice site mutation in neonatal carnitine palmitoyl transferase II deficiency

 Mitochondrial β-oxidation of long-chain fatty acids requires the concerted action of three tightly integrated membrane-bound enzymes (carnitine palmitoyltransferase I and II and carnitine/acylcarnitine translocase) that transport them into mitochondria. Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of this transport. We describe a novel splice-site mutation in the CPT II gene, found in a Moroccan family, of which four out of five children have died from the neonatal form of CPT II deficiency. Mutation detection studies at the mRNA level in the CPT II gene implied that the affected children were homozygous for the previously reported 534T insertion followed by a 25-bp deletion (encompassing bases 534–558). Studies of genomic DNA, however, revealed all patients to be compound heterozygous for this 534T ins/del 25 mutation, and for a new g→a splice-site mutation in the splice-acceptor site of intron 2. Because of these findings, prenatal diagnosis was performed in chorionic villi of three new pregnancies. This did not reveal new compound heterozygous genotypes, and, after uneventful pregnancies, all children appeared to be healthy. The new mutation is the first splice-site mutation ever identified in CPT II deficiency. The fact that it was not discovered in the patient's cDNA makes this study another example of the incompleteness of mutation detection at the mRNA level in cases where a mutation leads to aberrant splicing or nonsense-mediated messenger decay. Received: September 18, 2002 / Accepted: October 29, 2002 Acknowledgments We thank the patients and their family for participation, and Anouk Hanstede and Marloes Siers for technical assistance. Correspondence to:J.A.M. Smeitink     

左旋卡尼汀对缺氧/复氧诱导的心肌细胞氧化、凋亡影响的体外研究

目的：探讨左旋卡尼汀（L-carnitine）对缺氧/复氧（A/R）诱导的乳鼠心肌细胞抗氧化、抗凋亡效应及其可能的作用机制。方法： 采用原代培养乳鼠心肌细胞作为模拟缺血再灌注损伤实验模型，实验分3组：①正常对照组（control）；②A/R组:缺氧120 min,复氧240 min;③L-carnitine组：A/R前2 h加用L-carnitine设4个浓度梯度，1组20 mg/L，2组50 mg/L，3组100 mg/L，4组200 mg/L。观察各组细胞经A/R损伤后，细胞内超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量、琥珀酸脱氢酶（SDH）活性的变化情况，并用流式细胞仪（FCM）检测细胞凋亡率，透射电镜观察细胞超微结构的改变。结果： A/R组SOD活性、SDH活性明显低于对照组，MDA含量、细胞凋亡率均高于对照组(P＜0.05）；在L-carnitine用药组，随给药浓度的增加，MDA含量逐渐恢复正常，SOD活性、SDH活性逐渐回升，而且细胞凋亡率下降，各药物治疗组与A/R组比较各实验指标均显著差异(P＜0.05)；A/R组心肌细胞超微结构损伤明显重于药物治疗组。结论:左旋卡尼汀对心肌细胞具有保护作用，其作用在一定范围内呈剂量依赖关系。     

Formation of acetylcarnitine in muscle of horse during high intensity exercise

Summary To study the changes in carnitine in muscle with sprint exercise, two Thoroughbred horses performed two treadmill exercise tests. Biopsies of the middle gluteal were taken before, after exercise and after 12 min recovery. Resting mean muscle total carnitine content was 29.5 mmol · kg⁻¹ dry muscle (d. m.). Approximately 88% was free carnitine, 7% acetylcarnitine and acylcarnitine was estimated at 5%. Exercise did not affect total carnitine, but resulted in a marked fall in free carnitine and almost equivalent rise in acetylcarnitine. The results are consistent with a role for carnitine in the regulation of the acetyl-CoA/CoA ratio during sprint exercise in the Thoroughbred horse by buffering excess production of acetyl units.     

心肌损伤过程中肉毒硷与游离脂肪酸之间关系的探讨

本文观察了通过大鼠尾静脉给于内毒硷 (L-Carnitine,100mg/Lg/d、连续3日)对异丙基肾上腺素所致大鼠心肌损伤的影响。提示大鼠注射异丙基肾上腺素(ISP)后能显著降低心肌细胞内游离肉毒硷含量,同时使血中游离脂肪酸(FFA)含量明显升高,达正常值的4倍多。预先给大鼠注射 L-Carnitine,能阻止 ISP 降低心肌细胞游离肉毒硷的作用,并能显著降低血中 FFA 的水平,从而在一定程度上保护了心肌细胞。     

肉毒碱对兔在体缺血心室肌细胞动作电位的影响

本实验观察了人工合成肉毒碱d,1-carnitine盐酸盐(VBt)对在位兔心急性心肌缺血早期左室肌细胞动作电位的影响。阻断冠脉引起动作电位振幅(APA)和零期最大除极速度(dv/dt)明显降低以及复极50%时程(APD_(50))和复极90%时程(APD_(90))明显缩短。VBt有延长心室肌细胞动作电位时程的作用(P<0.01),并能显著减轻缺血心肌细胞的APD_(50)和APD_(90)的缩短程度,但对APA和dv/dt的缺血性变化则无明显改善。     

肝硬变时兔组织内肉毒碱含量的变化及对中、长链脂肪乳剂代谢的影响

21只新西兰兔经结扎胆总管制成肝硬变动物模型，而后随机分为胸外营养一ICT组（n＝10）及肠外营养一MCT/LCT组（n＝11），两组所含非蛋白热卡及氮量均相等，观察肝硬变时兔组织内肉毒碱含量的变化以及在肠外营养支持中应用ICT和MCT/后体内代谢的改变，并与正常对照组（n=10）比较。结果显示：肝硬变时兔血中甘油三酯、胆固醇及肉毒碱的含量均明显升高（P＜0．05），在场外营养第7天，LCT组甘油三脂、肉毒均高于MCT/LCT组（P＜o．01）；各组肝脏、心肌、骨骼肌及小肠中肉毒碱含量明显下降（P＜0．01）；肝内脂肪含量LCT组高于MCT/LCY组（P＜0．05）。本研究结果提示，MCT/LCT乳剂因其非肉毒碱依赖性，血中清除快，肝内脂肪存积少，可能是应用于肝硬变时更为理想的能源。     

Inhibition of carnitine synthesis modulates protein contents of the cardiac sarcoplasmic reticulum Ca2+-ATPase and hexokinase type I in rat hearts with myocardial infarction

It was previously reported than inhibition of carnitine synthesis by 3-(2,2,2-trimethyl-hydrazinium) propionate (MET-88) restores left ventricular (LV) systolic and diastolic function in rats with myocardial infarction (MI). Preservation of the calcium uptake function of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) is one of the possible mechanisms by which MET-88 alleviates hemodynamic dysfunction. To test this hypothesis, the effects of MET-88 on protein content of SERCA2 were evaluated using the same rat model of heart failure. Myocardial protein content of hexokinase, which is one of the key enzymes of glucose utilization, was also measured. Either MET-88 (MET-88 group) or a placebo (MI group) was administered for 20 days to rats with MI induced by coronary artery ligation. The control group underwent sham surgery (no ligation) and received placebo. In LV myocardial homogenates, the myocardial SERCA2 protein content was 32% lower (p<0.05) in the MI group than in the control group. However, in the MET-88 group myocardial SERCA2 content was the same as in the control group. Hexokinase I protein content was 29% lower (p<0.05) in the MI group compared with the control. In contrast, hexokinase II protein content did not differ significantly among the three groups. Consequently, inhibition of carnitine synthesis ameliorates depression of SERCA2 and hexokinase I protein content which may reduce tissue damage caused by MI. Received: 26 July 1999 Returned for 1. revision: 14 September 1999 1. Revision received: 14 December 1999 Returned for 2. revision: 26 January 2000 2. Revision received: 28 February 2000 Accepted: 6 April 2000     

Secondary Carnitine Deficiency in Handicapped Patients Receiving Valproic Acid and/or Elemental Diet

We examined serum-free carnitine (SFC) concentrations and serum acylcarnitine (SAC)/SFC ratios in 40 severely handicapped patients, aged 2 to 36 years, and 69 age-matched control subjects. SFC levels in the patients treated with valproic acid (VPA) and/or receiving carnitine-deficient elemental diets (ED) were significantly lower, and their SAC/SFC ratios were significantly higher than in the other patients or in control subjects. There were 6 patients whose SFC levels were less than the -2SD level (15.8 ± 6.7 μM, range 6.3±25.5) of those in control subjects (52.1±11.5 μM). They had no clinical symptoms of carnitine deficiency such as non-ketotic hypoglycemia, hepatomegaly, muscle weakness or cardiac function impairment, and showed normal transaminase, lipid and ammonia levels. In two cases (SFC=11.0, 13.4 μM), the ketogenic responses to intravenous administration of fat-emulsion were impaired, but they were restored after D-,L-carnitine supplementation (30mg/kg/day, po) for 1 month. However, in one case with the lowest SFC level (6.3 μM), the ketogenic responses to fat-emulsion infusion or fasting were normal, and dicarboxylic aciduria was not detected. These results indicate that 1) SFC levels are reduced in handicapped patients receiving VPA and/or ED, although clinical symptoms of carnitine deficiency do not easily develop, 2) some of these hypocarnitinemic cases show a subclinical impairment of hepatic fatty acid metabolism, not always correlated with the degree of SFC reduction, which can be restored by exogenous carnitine supplements, and therefore 3) in patients with acquired hypocarnitinemia, carnitine therapy should be considered, although a low SFC level alone may not imply an immediate indication.     

Lipid storage myopathies

Summary Various cases of lipid storage myopathies have been described. The biochemical defect could be determined in only some of these cases. The syndromes identified to date are as follows: carnitine deficiency (type I lipid storage myopathy), carnitine-palmityltransferase (CPT) deficiency and pyruvate-decarboxylase deficiency. In the last two diseases the vacuolization in muscle is not marked.The case of a 10 year old carnitine deficient patient with a history of insidious muscle weakness in the proximal limb and neck muscles is presented. The patient was treated with oral carnitine and a medium chain triglyceride diet for 18 months and her clinical status has remained improved. In other lipid storage patients prednisone treatment resulted in improvement.In cases of suspected lipid storage myopathy the following studies are indicated: 1) examination of ketone bodies in serum and urine during fasting, long chain and medium chain triglyceride diets; 2) serum triglyceride and serum carnitine; 3) study on fresh muscle and fibroblasts with labeled substrates, biochemical determination of carnitine and CPT in muscle.This paper was presented at the meeting of the Swiss Neurological Society and the Swiss Society of Muscular Disease in November 1975, Zürich, Switzerland     

抗氧化剂保护少弱精子离心过程氧化应激损伤的实验研究

目的：探讨抗氧化剂谷胱甘肽、左卡尼汀对少弱精子体外离心过程中抗氧化应激损伤的作用.方法：按WHO标准选取38份少弱精子样本,每份样本各取1350μL,分为对照组、谷胱甘肽组和左卡尼汀组,各450μL.对照组仅加入EBSS平衡液,谷胱甘肽组加入含有一定水平谷胱甘肽的EBSS平衡液,左卡尼汀组加入左卡尼汀的EBSS平衡液,检测三组活性氧（ROS）、丙二醛（MDA）及精子DNA断裂指数（DFI）并进行比较、分析.结果：三组组间比较均有统计学意义（F活性氧=9.45、P=0.000;F丙二醛=15.79,P=0.000;FDFI=13.56,P=0.000,P均〈0.05）;两两比较示谷胱甘肽组与左卡尼汀组均较对照组间的活性氧、丙二醛及DFI水平明显降低,差异有统计学意义（P均〈0.05））;而谷胱甘肽组及左卡尼汀组间的比较均无统计学意义（P均〉0.05）.结论：在精液离心前添加一定浓度的左卡尼汀或谷胱甘肽可减少离心过程中产生的过量活性氧对精子的氧化应激性损伤,从而提高精子质量.