GC-MS Investigation of Metabolic Activity in 4-Hydroxy-2-Oxoglutarate Aldolase Deficiency of Primary Hyperoxaluria Type 3

Primary hyperoxaluria (PH) is a group of three variants of an inborn error of glyoxylate metabolism. The hallmark of PH is the overproduction of oxalate which is formed from oxidation of glyoxylate by lactate dehydrogenase (LDH) and which readily crystallizes as calcium oxalate in renal tissue. PH3 is the most recently characterized of the variants of PH, and it is also the least understood. PH3 is caused by a malfunction in the enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA), which catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to form glyoxylate and pyruvate. Since the enzymatic malfunction occurs before glyoxylate formation, and previous studies have shown HOG to be elevated in the urine of PH3 patients, it is unclear how the phenotype of PH3 manifests. If HOG can be directly excreted before glyoxylate is ever formed, it is difficult to imagine how a disease state occurs. It has been proposed that HOG, upon exceeding normal cellular levels, must flux out of the mitochondria where it is normally metabolized and be acted upon by another enzyme. It is unclear whether HOG is either metabolized by another aldolase, some other type of enzyme or metabolized after it has undergone some other chemical modification, as indicated by the presence of the reduced product of HOG, 2,4-dihydroxyglutarate (DHG) in the urine of PH3 patients. The goal of this thesis was to develop a method to analyze PH3 metabolic consequences in liver tissue lysates and urine from wild type (WT) and HOGA knockout (KO) mice in order to indicate a plausible direction for future research into the consequences of this disease. After extensive optimization, a successful GC-MS analytical method was established, and preliminary qualitative results were obtained from WT and KO liver and urine. In liver several qualitative metabolic differences were detected, and oxoadipic acid was detected in all of the samples; importantly it did not interfere with HOG detection. DHG was identified from HOGA KO urine and incorporated into the in-house spectral library. Two unknowns were identified in liver as dihydrouracil and phosphate, and two more unknowns were detected but not yet identified in urine.

Grantor dc:publisher

Wake Forest University

Year dc:date.issued

2016

Author dc:creator

• Everett, Jordan

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10339/62654

OAI identifier oai:identifier

oai:wakespace.lib.wfu.edu:10339/62654