BCH 545 Lecture 2 2025 PDF

Inborn errors of pyruvate and TCA metabolism Pro f. A b i r G h a nnouc hi BCH 5 4 5 2 0 2 4- 2025  Owing to the role of pyruvate and the TCA cycle in energy metabolism, as well as in gluconeogenesis, lipogenesis and AA synthesis, defects in pyruvate metabolism and in t...

Inborn errors of pyruvate and TCA metabolism Pro f. A b i r G h a nnouc hi BCH 5 4 5 2 0 2 4- 2025  Owing to the role of pyruvate and the TCA cycle in energy metabolism, as well as in gluconeogenesis, lipogenesis and AA synthesis, defects in pyruvate metabolism and in the TCA cycle almost invariably affect the CNS. PC Deficiency is caused by mutations in the PC gene, which is a gene that provides the instructions for creating an enzyme known as PC. Pyruvate Carboxylase Gene Pyruvate Carboxylase Enzyme How does it get passed down? Pyruvate Carboxylase deficiency is autosomal recessive Mother Carrier Father Carrier This means when both the mother and father are carriers of the mutated gene that causes this disorder, their future child has a 25% chance of getting this disorder. The PC enzyme is used in the mitochondria in the Krebs cycle to catalyze the chemical reaction that converts a pyruvate molecule into a molecule known as oxaloacetate. This reaction is essential, as oxaloacetate is used in the Krebs cycle which is a process that contributes towards PC deficiency results in lower energy production for the amounts oxaloacetate body. molecules being produced. This restricts the Krebs cycle from taking place and causes the body to be deprived of energy.  With PC deficiency, there is less glucose generation, resulting in the body also being deprived of energy. Gluconeogenesis generates glucose (the body’s main energy source), when carbohydrate intake is low. PC catalyzes the conversion of pyruvate molecules into oxaloacetate in the first step of gluconeogenesis in the kidneys and liver.  Lactic Acid Build Up Does not occur Lactate Dehydrogenase Occurs instead O O− O O− C NADH + H+ NAD+ C C O HC OH LACTIC ACID BUILDUP LEADS CH3 CH3 TO LACTIC ACIDOSIS!! pyruvate lactate Effect on Nervous System Neurotransmitters In the nervous system, PC has the important role of replenishing certain components needed for making neurotransmitters that are used for nerve cell communication→ This means that a deficiency in this enzyme affects the production of neurotransmitters which can cause developmental delays. PC also has an important role in the creation of myelin, a protective sheath that surrounds nerve cells. Having PC deficiency can negatively affect nerve cells which can also lead to developmental delays. Myelin Sheath Symptoms Developmental delay Deprivation of energy Lactic acidosis Diagnosis Molecular Genetic Testing Identifying Pyruvate Carboxylase enzyme in fibroblasts 5% 25% Viewing Symptoms Type A Type B Type C Treatments As of now, no complete cure for PC deficiency has been discovered. Treatment of this disorder is aimed to provide alternative sources of energy for the body by alternative methods to metabolizing pyruvate. COO- NAD+ NADH + H + O C O + HSCoA H3C C ~SCoA + CO2 Pyruvate CH3 dehydrogenase complex pyruvate Acetyl CoA  Lactate Dehydrogenase O O− O O− + + C NADH + H NAD C C O HC OH CH3 CH3 pyruvate lactate COO - NAD+ NADH + H + O C O + HSCoA H3C C ~SCoA + CO2 Pyruvate CH3 dehydrogenase complex pyruvate Acetyl CoA As pyruvate metabolism increases through using this technique, pyruvate and lactate levels decreases which overall decreases the amount of lactic acid present in the blood Biotin supplementation Biotin is a cofactor of PC and taking biotin supplements will increase enzyme activity, but it is usually of little use except potentially in a mildly affected type C patient. Citrate Supplementation PEP Carboxykinase (PEPCK) Deficiency Patients reported to be PEPCK deficient presented, as those with PC deficiency, with acute episodes of severe lactic acidosis associated with hypoglycemia. Onset of symptoms is neonatal or after a few months. Patients display mostly progressive multisystem damage with failure to thrive, muscular weakness and hypotonia, developmental delay with seizures, spasticity, microcephaly. Phosphoenolpyruvate Carboxykinase (PEPCK) Deficiency Hepatomegaly with hepatocellular dysfunction Renal tubular acidosis Cardiomyopathy PEPCK presents as 2 isoforms, mitochondrial and cytosolic, which are encoded by two distinct genes The deficiency of mitochondrial PEPCK, which intervenes in gluconeogenesis from lactate, should have more severe consequences. Cytosolic PEPCK, which is supposed to play a role in gluconeogenesis from alanine. Diagnostic Tests The diagnosis of PEPCK deficiency is complicated by the existence of separate mitochondrial and cytosolic isoforms of the enzyme. Optimally, both isoforms should be assayed in a fresh liver sample after fractionation of mitochondria and cytosol. In cultured fibroblasts, most of the PEPCK activity is located in the mitochondrial compartment, and low PEPCK activity in whole-cell homogenates indicates deficiency of the mitochondrial isoform. Treatment and Prognosis Patients with suspected PEPCK deficiency should be treated with intravenous glucose and sodium bicarbonate during acute episodes of hypoglycemia and lactic acidosis. Fasting should be avoided, and cornstarch or other forms of slow-release carbohydrates need to be provided before bedtime. The long-term prognosis of patients with reported PEPCK deficiency is usually poor, with most subjects dying of intractable hypoglycemia or neurodegenerative disease.  COO - NAD+ NADH + H + O C O + HSCoA H3C C ~SCoA + CO2 Pyruvate CH3 dehydrogenase complex pyruvate Acetyl CoA Thiamine pyrophosphate, TPP (VB1) HSCoA (pantothenic acid) Cofactors lipoic Acid NAD+ HSCoA FAD (VB2) NAD+  GLUCOSE Input to Krebs Cycle, where the acetate moiety is further glycolysis degraded to CO2. Donor of acetate for synthesis PYRUVATE of fatty acids, ketone bodies, & cholesterol. Pyruvate dehydrogenase lipogenesis Acetyl CoA Fatty acids citric acid -oxidation (Cytoplasm) cycle ketone cholesterol CO2 ketogenesis (liver only) oxidation synthesis Ketone bodies steroid Cholesterol hormones (endocrine glands) Metabolic sources and fates of acetyl CoA Metabolic Derangement Defects of PDHC provoke conversion of pyruvate into lactate rather than in acetyl- CoA, the gateway for complete oxidation of carbohydrate via the TCA cycle. Only 2 ATP is produced instead of 36-38 ATP. Cytosol Mitochondrion Glycolysis 2 2 Krebs Acetyl- Electron Glucose Pyruvic Cycle CoA Transport acid Maximum per glucose: by direct by by synthesis direct ATP synthesis synthase Metabolic Derangement Deficiency of PDHC thus specifically interferes with production of energy from carbohydrate oxidation, and lactic acidemia is aggravated by consumption of carbohydrate. Clinical presentation Neurological: hypotonia, weakness, ataxia, spasticity, cerebellar degeneration, seizure, mental retardation Brain malformations: microcephaly, narrowed head,wide nasal bridge, wide eye-corner (look like and being a debil) Lactic acidosis: pyruvate is increased in blood, pH lowered Clinical presentation If pyruvate dehydrogenase or dihydrolipoate transacetylase (E2) or PDHC phosphatase (E1) is deficient, only this enzyme complex is affected. If dihydrolipoyl dehydrogenase, the 3rd subunit is missing, the complete deficiency is fatal in utero, the partial defect is untreatable.  E3 subunit is the same in α- ketoglutarate dehydrogenase and branched chain α -ketoacid dehydrogenase complex, consequently neither citric acid cycle, the common degradative process of carbohydrates, lipids and amino acids can work, nor branched chain amino acids can be broken down → In every cell always just anaerobic glycolysis can proceed. Serious developmental irreversible organic and functional brain damage occurs in utero, during and after birth because: Brain has extraordinary high energy demand to maintain the ionic concentration after action potential and signal transduction. 20 % of O2 is consumed by adult brain in human body (60 % in child) 60-70 % of all ATP is used by Na-K-ATPase in brain. It needs maximal activity of PDHC. Anaerobic glycolysis can not be accelerated enough to compensate its inefficient ATP production. Acetylcholine neurotransmitter synthesis requires acetyl-CoA, produced by PDHC. Production of glutamate, GABA, aspartate neurotransmitters needs the entrance of acetyl-CoA to citric acid cycle. PDC deficiency and brain damage Missing any of the coenzymes causes acquired enzyme deficiency: mainly thiamin, riboflavin and niacin deficiency occurs in alcoholics and in serious starvation. PDC deficiency and brain damage Neurons use only glucose to fulfill their energy demand, Fatty acids can not penetrate through BBB and degradation of their own sythesized FA is slow Ketone body production is significant just after several days starvation (then they are used instead of more than 50 % of glucose). AAs degradation is not significant normally, but neurons consume AAs instead of synthesizing proteins in this case Other organs can degrade fatty acids efficiently, therefore they are not damaged. Treatment and Prognosis The general prognosis for individuals with PDHC deficiency is poor, and treatment is not very effective. Experience with early prospective treatment to prevent irreversible brain injury is lacking. Perhaps the most rational strategy for treating PDHC deficiency is the use of a ketogenic diet. Oxidation of fatty acids, 3-hydroxybutyrate, and acetoacetate are providers of alternative sources of acetyl-CoA. Laboratory diagnosis The most important laboratory test for initial recognition of PDHC deficiency is measurement of blood and CSF lactate and pyruvate. Quantitative analysis of plasma amino acids and urinary organic acids may also be useful. Blood lactate, pyruvate and alanine can be intermittently normal, but, characteristically, an increase is observed after an oral carbohydrate load. Laboratory diagnosis While L/P ratio is as a rule normal, a high ratio can be found if the patient is acutely ill, if blood is very difficult to obtain, or if the measurement of pyruvate (which is unstable) is not done reliably. The practical solution to avoid these artifacts is to obtain several samples of blood, including samples collected under different dietary conditions (during an acute illness, after overnight fasting, and postprandially after a high- carbohydrate meal). Laboratory diagnosis Glucose-tolerance or carbohydrate-loading tests are not necessary for a definite diagnosis. In contrast to deficiencies of PC or PEPCK, fasting hypoglycemia is not an expected feature of PDHC deficiency. Ketoglutarate Dehydrogenase Complex (KDHC) Deficiency Since KDHC is integral to the TCA cycle, its deficiency has consequences similar to that of other TCA enzyme deficiencies. Diagnostic Tests The most useful test for recognizing KDHC deficiency is urine organic acid analysis, which can show increased excretion of α-KGA with or without concomitantly increased excretion of other TCA cycle intermediates. However, mildly to moderately increased urinary α-KGA is a common finding and not a specific marker of KDHC deficiency. Diagnostic Tests Some patients with KDHC deficiency also have increased blood lactate with normal or increased L/P ratio. Plasma glutamate and glutamine may be increased. KDHC activity can be assayed through the release of 14CO2 from 2-ketoglutarate in crude homogenates of cultured skin fibroblasts, muscle homogenates and other cells and tissues Fumarase Deficiency All patients had a profound mental retardation and presented as a static encephalopathy. Six out of 8 developed seizures. The seizures were of various types and of variable severity, but several patients experienced episodes of status epilepticus. Decreased white matter, and a small brainstem are considered characteristic Fumarase Deficiency Its deficiency, like other TCA cycle defects, causes: impaired energy production caused by interrupting the flow of the TCA cycle potential secondary enzyme inhibition associated with accumulation in various amounts of metabolites proximal to the enzyme deficiency such as fumarate, succinate, 2-KGA and citrate Treatment and Prognosis There is no specific treatment. While removal of certain amino acids that are precursors of fumarate could be beneficial. Conversely, supplementation with aspartate or citrate might lead to overproduction of toxic TCA cycle intermediates.  Succinate Dehydrogenase (SD) Deficiency SD is part of both the TCA cycle and the respiratory chain. This explains why SD deficiency resembles more the phenotypes associated with defects of the respiratory chain. Succinate Dehydrogenase (SD) Deficiency The clinical picture of this very rare disorder can include: Isolated hypertrophic cardiomyopathy, combined cardiac and skeletal myopathy, generalized muscle weakness with easy fatigability. It can also present with optic atrophy and tumor formation in adulthood. Profound hypoglycemia was seen in some infants. Succinate Dehydrogenase (SD) Deficiency SD deficiency may also present as a compound deficiency state that involves aconitase and complexes I and III of the respiratory chain. This disorder, found only in Swedish patients, presents with life-long exercise intolerance, myoglobinuria, and lactic acidosis. Metabolic Derangement Theoretically, TCA-cycle defects should lead to a decreased L/P ratio, because of impaired production of NADH. However, too few cases of SD deficiency (or other TCA-cycle defects) have been evaluated to determine whether this is a consistent finding. Profound hypoglycemia, as reported once, might have resulted from the depletion of the gluconeogenesis substrate, oxaloacetate. The combined SD/aconitase deficiency found only in Swedish patients, appears to be caused by a defect in the metabolism of the iron-sulfur clusters common to these enzymes. Diagnosis In contrast to the other TCA cycle disorders, SD deficiency does not always lead to a characteristic organic aciduria. Many patients, especially those whose clinical phenotypes resemble the patients with respiratory chain defects, do not exhibit the expected succinic aciduria and can excrete variable amounts of lactate, pyruvate, and the TCA cycle intermediates, fumarate and malate. Diagnostic confirmation of a suspected SD deficiency requires analysis of SD activity itself, as well as complex- II(succinate-ubiquinone oxidoreductase) activity, which reflects the integrity of SD and the remaining two subunits. Pyruvate Transporter Defect Only one patient has been completely documented. Neonatal lactic acidosis in a female baby from consanguineous parents was associated with generalized hypotonia and facial dysmorphism. MRI of the brain revealed cortical atrophy, periventricular leukomalacia and calcifications. Progressive microcephaly, failure to thrive and neurological deterioration led to death at the age of 19 months.