FUNGAL GLUCOAMYLASE SUPPLEMENTS ON STARCH DIGESTION IN GLUCOSIDASE DEFICIENT

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 摘要淀粉和糖占人类膳食能量摄入量的60%-90%。所有食物碳水化合物(CHO)S在被称为双糖酶的膜结合酶家族吸收之前在小肠中被消化为单糖。研究最多的双糖酶是乳糖酶和蔗糖酶-异麦芽糖酶。先天性蔗糖酶异麦芽糖酶缺乏症(CSID)由于临床症状明显，已被从临床、蛋白质组和基因组水平进行了研究。虽然唾液和胰腺α-淀粉酶活性在淀粉消化中的作用是众所周知的，但粘膜α-葡萄糖苷酶活性的作用却知之甚少。α-淀粉酶只释放支链淀粉中葡萄糖的4%。其余96%的产物是具有保守长度模式的可溶性葡萄糖低聚物。这些低聚物，包括麦芽糖(G2)和通过G40的长度需要进一步消化。粘膜葡萄糖苷酶从可溶低聚物的非还原末端释放游离葡萄糖。1880年首次报道了淀粉酶和葡萄糖苷酶活性的区别，当时发现了小肠中的麦芽糖酶活性，而胰腺中没有麦芽糖酶活性。20世纪60年代，Dahlqvist发现在人的空肠中存在4种不同的麦芽糖酶活性，其中两种具有蔗糖-异麦芽糖酶(SI)活性，另两种不具有其他所有二糖酶活性，称为麦芽糖酶-糖淀粉酶(MGAM)。20世纪80年代，Hermann等人提出。证明了粘膜麦芽糖酶活性是葡萄糖苷酶的一个亚类，当SI和MGAM的底物特异性相同时，MGAM的活性是100倍，SI肽的浓度是20倍。这就要求必须对两者进行研究，以了解从食物淀粉中产生的粘膜葡萄糖。 该方案是H-20932的通用版本，该方案是为一个遗传性淀粉消化不良的家庭编写的。在这个方案中，我们将研究一个全国样本，这些儿童的十二指肠活检麦芽糖酶活性低，蔗糖酶活性正常。这项研究的动力与两个年轻的兄弟姐妹有关，他们被认为是淀粉不耐受的继发性疾病。他们寻求治疗这种目前由饮食限制管理的疾病(见H-20932)。在年龄较大的孩子中，这种情况被归因于经活检证实的先天性缺乏称为麦芽糖酶的肠道刷状边界酶。活检组织的葡萄糖苷酶活性分别为蔗糖酶40.6(NL25)、麦芽糖酶87.1(NL100)和软脂酶6.8(NL5)。所有数值均为um/min/g蛋白质。 葡萄糖苷酶将低聚糖还原为可被人体吸收的单糖。失败会导致未被消化的复杂碳水化合物进入结肠，并引发气体、腹胀、易怒和腹泻。1994年，Lebenthal描述了一个类似的综合征，9名6-107个月大的儿童被发现患有临床淀粉不耐受。莱本塔尔使用糖原作为粘膜活检葡萄糖苷酶检测的底物，并报告活性不足，这与他的临床诊断基本一致。我们现在知道糖原底物只有大约80%是粘膜MGAM活性的特异性。在Lebenthals论文发表以来的14年里，我们已经证明了四种葡萄糖苷酶参与了淀粉酶后将低聚糖消化为葡萄糖，而特定的底物不能分解这些葡萄糖苷酶的活性。我们已经发现，免疫沉淀活性可以解决不同基因对淀粉消化的个体贡献。 假设补充含有真菌淀粉糖苷酶的口服酶疗法将有益于有症状的先天性麦芽糖酶缺乏症儿童，表现为症状改善和呼吸样本富集值增加(目标替代刷状边界α-极限糊精消化和产物同化)。 一、具体目标 1.证明含真菌淀粉糖苷酶的补充口服酶疗法对有症状的先天性麦芽糖酶缺乏症患儿是有益的。 2.证明症状有所改善 3.证明呼吸样本富集值增加(刷状边缘α-极限-糊精消化和产物同化的客观替代物)

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABSTRACT Starches and sugars make up 60-90% of human dietary energy intakes. All food carbohydrates (CHO)s are digested in the small intestine to monosaccharides before absorption by a family of membrane bound luminal enzymes called disaccharidases. The best studied disaccharidases are lactase, and sucrase-isomaltase. Because of recognizable clinical symptoms, congenital sucrase-isomaltase deficiencies (CSID) have been studied at clinical, proteomic and genomic levels. While the role of salivary and pancreatic alpha-amylase activities in starch digestion are well known, the roles of mucosal alpha-glucosidases activities are less understood. Alpha-amylases release only 4% of the glucose present in amylopectin. The remaining 96% of products are soluble glucose oligomers with a conserved pattern of lengths. These oligomers, including maltose (G2) and lengths through G40 require further digestion. Mucosal glucosidases release free glucose from the non-reducing ends of the soluble oligomers. Differentiation of amylase from glucosidases activity was first reported in 1880 after discovery of maltase activity in small intestine and its absence in the pancreas. In the 1960s Dahlqvist found that 4 different maltase activities could be identified in the human jejunum; two were isolated with sucrase-isomaltase (SI) activities and two, that were free of all other disaccharidase activities, were called maltase-glucoamylase (MGAM). In the 1980s Hermann, et al. proved that mucosal maltase activities are a subgroup of the glucosidases and that while substrate specificities of SI and MGAM are identical, the activity of MGAM was 100 fold greater and the concentration of SI peptide is 20 fold greater. This dictates that both must be investigated to understand mucosal glucose production from food starches. This protocol is a generic version of H-20932 which was written for a family with hereditary poor starch digestion. In this protocol we will study a national sample of children with similar low duodenal biopsy maltase and normal sucrase activities. The impetus for this study relates to two young siblings with failure-to-thrive that is believed to be secondary to starch intolerance. They have sought treatment for this condition that is currently managed by dietary restrictions (see H-20932). In the older child the condition has been attributed to biopsy-proven, congenital absence of the intestinal brush border enzyme called maltase. The glucosidase activities for the biopsy were, sucrase 40.6 (nl 25), maltase 87.1 (nl 100), and palatinase 6.8 (nl 5). All values were uM/min/g protein. Glucosidase enzymes reduce oligosaccharides to simple glucose that can be absorbed into the body. Failure results in undigested complex carbohydrates that pass to the colon and provoke gas, bloating, irritability and diarrhea. A similar syndrome was described by Lebenthal in 1994, nine children aged from 6-107 months were found to have clinical starch intolerance. Lebenthal used glycogen as substrate for a mucosal biopsy glucosidase assay and reported that deficient activity generally agreed with his clinical diagnosis. We now know that glycogen substrate is only about 80% specific for the MGAM activity of the mucosa. In the 14 years since Lebenthals paper we have shown that four glucosidases participate in the post-amylase digestion of oligosaccharides to glucose and that specific substrates cannot dissect these glucosidase activities. We have found that immunoprecipitation of the activities can resolve individual contributions by the different genes to starch digestion. HYPOTHESIS Supplemental oral enzyme therapy, containing fungal amyloglucosidase, will be beneficial to symptomatic children with congenital maltase glucoamylase deficiency as shown by symptom improvement and increased breath sample enrichment values (objective surrogate of brush border alpha-limit-dextrin digestion and product assimilation) . I. SPECIFIC AIMS 1. To demonstrate that supplemental oral enzyme therapy, containing fungal amyloglucosidase, is beneficial to symptomatic children with congenital maltase glucoamylase deficiency. 2. To demonstrate symptom improvement 3. To demonstrate increased breath sample enrichment values (objective surrogate of brush border alpha-limit-dextrin digestion and product assimilation)