Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, UFRGS, Av. Ipiranga, 27522, CEP, 90610-000, Porto Alegre, RS, Brazil; Serviço de Genética Médica, HCPA, R.Ramiro Barcelos, 2350, CEP, 90035-003, Porto Alegre, RS, Brazil.
Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, UFRGS, Av. Ipiranga, 27522, CEP, 90610-000, Porto Alegre, RS, Brazil; Serviço de Genética Médica, HCPA, R.Ramiro Barcelos, 2350, CEP, 90035-003, Porto Alegre, RS, Brazil.
Arch Biochem Biophys. 2019 Jun 15;668:16-22. doi: 10.1016/j.abb.2019.04.008. Epub 2019 Apr 30.
3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 μM and 150 μM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.
3-羟基-3-甲基戊二酸尿症（HMGA）是亮氨酸分解代谢途径中的一种遗传性疾病，其中 3-羟基-3-甲基戊二酰辅酶 A 裂解酶缺乏。因此，有机酸酸 3-羟基-3-甲基戊二酸（HMG）和 3-甲基戊二酸（MGA）主要在受影响患者的组织中积累。最近，人们越来越关注自由基作为人类疾病中组织损伤的介质，导致脂质过氧化、蛋白质氧化和 DNA 损伤。这种疾病的治疗基于限制蛋白质摄入和补充抗氧化剂和解毒剂左旋肉碱（LC）。在本工作中，我们研究了有机酸酸积累诱导的体外 DNA 氧化损伤和 3-HMG 患者体内的氧化应激参数，以及推荐治疗的效果。通过在白细胞中孵育 HMG 和 MGA（1mM、2.5mM 和 5mM）并与 LC（90μM 和 150μM）共孵育，用碱性彗星试验分析体外 DNA 损伤。在 LC 治疗前后，用 ELISA 试剂盒测量患者尿液中的尿 15-F2t-异前列腺素水平和尿氧化鸟嘌呤。HMG 和 MGA 诱导的 DNA 损伤指数（DI）明显高于对照组。在 LC 的存在下，DI 显著降低。还验证了氧化鸟嘌呤和尿液异前列腺素水平分别显著升高，这是氧化 DNA 损伤和脂质过氧化的生物标志物，在治疗前的患者中。LC 治疗和补充后，患者的这些生物标志物水平显著降低。综合分析数据，我们可以得出结论，HMGA 患者存在由 HMG 和 MGA 诱导的氧化脂质和 DNA 损伤，而 LC 的抗氧化治疗可以预防这种损伤。
