Ashadi Sasongko Essential facts of the citric acid cycle • The citric acid cycle is an eight-step reaction. • It requires 8 enzymes. • The final product is oxaloacetate. • Three NADH molecules are produced. • One GTP molecule is produced. • One FADH2 molecule is produced. • The cycle is accompanied by the liberation of 2 CO2 molecules. Eight enzymes utilized in the citric acid cycle 1. Citrate synthase 2. Aconitase 3. Isocitrate dehydrogenase 4. a-Ketoglutarate dehydrogenase 5. Succinyl-CoA synthetase 6. Succinate dehydrogenase 7. Fumarase 8. Malate dehydrogenase Step 1: Acetyl-CoA Citrate In the first step of the cycle, the enzyme citrate synthase catalyzes the condensation of acetylCoA with oxaloacetate. Step 2: Citrate Isocitrate In step 2 of the cycle, there are two substeps used to generate isocitrate, which is easier to oxidize. Step 3: Isocitrate α-Ketoglutarate The next step in the citric acid cycle also has two substeps or phases. First, isocitrate is oxidized by the enzyme isocitrate dehydrogenase producing oxalosuccinate. Like cis-aconitate in step 2, this molecule is an intermediate that never dissociates from the enzyme. Step 4: α-Ketoglutarate Succinyl-CoA The next step in the reaction, which is catalyzed by a-ketoglutarate dehydrogenase, decarboxylates a-ketoglutarate producing succinyl-CoA. Step 5: Succinyl-CoA Succinate In this step, a high energy GTP molecule is produced. This is substrate level phosphorylation. The reaction is catalyzed by the succinyl-CoA synthetase, and the coenzyme-A molecule consumed in the production of succinyl-CoA is released. Step 6: Succinate Fumarate In this step, succcinate dehydrogenase oxidizes the succinate molecule producing fumarate. Step 7: Fumarate L-Malate In this step, fumarate hydratase or fumarase catalyzes a reversible reaction in which fumarate is transformed into malate. Step 8: Malate Oxaloacetate In this last step, the molecule oxaloacetate is regenerated from malate. The enzyme which catalyzes this step is malate dehydrogenase, and 1 NADH molecule is produced What is Metabolomics? What is Metabonomics? The ensemble of metabolites in an organism is known as its metabolome. In 1999 the term metabonomics was devised to describe “the multiparametric, quantitative study of dynamic metabolome responses in living systems to physiological and pathophysiological stimulation or genetic modification”. However, a competing term appeared in 2001: metabolomics, that was generally defined as: the “comprehensive and quantitative analysis of all metabolites…” The urine samples obtained from heat stressed rats contained altered concentration of citrate (2.66), succinate ( 2.42), 2-oxoglutrate ( 2.98), phenylalanine ( 3.26, 4.06, 7.30, 7.34), creatinine ( 3.02, 4.06), hippurate (7.54, 7.82) and formate ( 8.46) in comparison with control rats The urine samples obtained from heat stressed rats contained altered concentration of citrate (2.66), succinate ( 2.42), 2-oxoglutrate ( 2.98), phenylalanine ( 3.26, 4.06, 7.30, 7.34), creatinine ( 3.02, 4.06), hippurate (7.54, 7.82) and formate ( 8.46) in comparison with control rats The results of the present study showed significant effects on the metabolites involved in several pathways such as tricarboxylic acid (TCA) cycle (citrate, 2-oxoglutrate, succinate) The urinary excretion levels of citrate, 2-oxoglutrate, and succinate were decreased. As reported earlier, irrespective of any kind of physiological stress, there is increased energy consumption and protection against internal and external stress is provided by allostatis. During heat exposure, increased energy consumption is expected. However, in our studies the decrease in TCA cycle metabolites can be explained by two stages. Initially, during heat exposure, TCA cycle is accelerated due to enhanced adrenergic nerve activity. As soon as the rats are returned back to metabolic cages, they appeared less active indicating initiation of recovery process and thus slower energy consumption period. The short-term exposure to acute heat stress followed by long room temperature recovery process leads to overall lower level of TCA cycle metabolites in twelve hours urine sample. Hence, alteration of the TCA cycle is an important part of metabolic regulatory and compensatory mechanism in response to heat stress exposure. conclusion NMR based metabonomic studies in conjugation with statistical analysis permits non-invasive and simultaneous monitoring of various metabolic pathways revealing a subtle interplay of functional metabolites and pathways leading to an understanding of the systemic response to external stimuli such as heat stress. The results provides a new insight into the changes induced by heat stress at metabolic level showing a decrease in metabolites involved in TCA cycle (succinate, 2-oxoglutrate and citrate) and catecholamine metabolic pathway (phenylalanine).