Endogenous Oxalate Synthesis

描述：内源性草酸合成在特发性草酸钙结石和原发性高草酸尿症中起重要作用。尽管进行了数十年的研究，但人们对这种合成的基础途径知之甚少。在上一个资金周期中，我们确定了氨基酸和糖的代谢，以前被认为是主要的贡献，但只产生有限数量的草酸。在这一应用中，我们建议调查乙二醛是否是草酸合成的重要来源。我们的初步研究表明乙二醛转化为草酸。乙二醛主要通过与谷胱甘肽和乙二醛酶系统的相互作用转化为乙醇酸。这些通路的活性取决于谷胱甘肽和NADPH。任何降低这些成分的氧化胁迫都有可能加速草酸的合成。已经制定了三个具体目标。具体目标1：利用纯化的重组酶和siRNA敲除培养的肝癌细胞，对可能催化乙二醛转化为草酸的酶(S)进行评估。乙二醛生成和乙二醛酶系统的相互关系将通过谷胱甘肽耗竭来检验。葡萄糖和亚油酸将被评估为利用人类红细胞和HepG2细胞产生乙二醛和草酸的潜在来源。使用~(13)C-同位素和~(13)C标记的草酸、乙醇酸和乙二醛的离子色谱与质控联用(IC/MS)或液-质联用联用(LC/MS)将使碳的通量能够被跟踪。具体目标2将评估修改乙醛酶系统是否影响草酸合成。据推测，减少这个体系将增加草酸盐的合成。人红细胞、乙二醇酶-1(GLO-1)缺陷小鼠和野生型小鼠的红细胞将暴露在乙二醛、谷胱甘肽耗竭和氧化应激中。预计这些动作将增加草酸的合成，特别是在缺乏GLO-1的细胞中。HepG2细胞、GLO-1缺陷和野生型小鼠也将受到氧化应激、谷胱甘肽耗竭和抗氧化剂的影响，以评估乙醛酸酶系统的影响。在具体目标3中，将评估关于2型糖尿病与内源性草酸合成增加有关的假设。正常成年人、患有和不伴有2型糖尿病的草酸钙结石的人，以及没有肾结石病史的糖尿病患者将被描述为特征。对内源性草酸合成的贡献将通过让参与者服用严格控制的低草酸饮食来估计。尿草酸和乙醇酸排泄与氧化应激和乙二醛产生的关系将被确定。Zucker大鼠是一种患有胰岛素抵抗的动物，最近发现草酸排泄增加，将被用来确定抗氧化治疗是否可以减少乙二醛的产生以及尿中的乙醇酸和草酸。拟议的实验应该对草酸盐是如何合成的产生新的见解，并应该揭示抗氧化疗法是否是防止草酸钙结石形成的一种潜在疗法。

DESCRIPTION: Endogenous oxalate synthesis plays an important role in idiopathic calcium oxalate stone disease and primary hyperoxaluria. The pathways that underlie this synthesis are poorly understood despite decades of research. In the last funding cycle we established that amino acid and sugar metabolism, previously believed to make major contributions, produce only a limited amount of oxalate. In this application, we propose investigating whether glyoxal is a significant source of oxalate synthesis. Our preliminary studies have shown that glyoxal is converted to oxalate. Glyoxal is primarily converted to glycolate by its interaction with glutathione and the glyoxalase system. Activity of these pathways is contingent on glutathione and NADPH. Any oxidant stress reducing these components could potentially accelerate oxalate synthesis. Three specific aims have been developed. Specific Aim 1: The enzyme(s) potentially catalyzing the conversion of glyoxal to oxalate will be assessed using purified recombinant enzymes and siRNA knockdown in cultured hepatoma (HepG2) cells. The inter-relationship with glyoxal generation and the glyoxalase system will be examined using glutathione depletion. Glucose and linoleate will be assessed as potential sources of glyoxal and oxalate generation using human red cells and HepG2 cells. The use of 13C-isotopes and the quantification of 13C-labelled oxalate, glycolate and glyoxal with ion chromatography coupled to mass detection (IC/MS) or liquid chromatography coupled to mass detection (LC/MS), will allow the flux of carbon to be tracked. Specific Aim 2 will assess if modifying the glyoxalase system impacts oxalate synthesis. It is hypothesized that reducing this system will increase oxalate synthesis. Human erythrocytes and those from Glyoxalase-1 (GLO-1) deficient and wild type mice will be exposed to glyoxal, glutathione depletion and oxidative stress. It is anticipated that these maneuvers will increase oxalate synthesis especially in GLO-1 deficient cells. HepG2 cells, GLO-1 deficient and wild type mice will also be subjected to oxidative stress, glutathione depletion and antioxidants to assess the impact of the glyoxalase system. In Specific Aim 3 the hypothesis that type 2 diabetes is associated with increased endogenous oxalate synthesis will be assessed. Normal adults, those with calcium oxalate stones with and without type 2 diabetes, and diabetics without a history of kidney stones will be characterized. The contribution to endogenous oxalate synthesis will be estimated by placing the participants on tightly controlled low oxalate diets. The relationships between urinary oxalate and glycolate excretion to oxidative stress and glyoxal production will be determined. The Zucker rat, an animal with insulin resistance and recently identified increased oxalate excretion, will be utilized to determine if antioxidant therapy can decrease glyoxal production and urinary glycolate and oxalate. The proposed experiments should produce novel insights into how oxalate is synthesized and should reveal whether antioxidant therapy is a potential therapy to prevent calcium oxalate stone formation.