Lactate and Enzymes - Essay Example

Lactate & Enzymes Under what conditions, and why is lactate produced in the body Occurring naturally, lactate is a compound formed in the body. These are by-products of and energy for exercise: which is commonly found in the muscles, the blood and various organs. The primary source of lactate is the breakdown of stored carbohydrate called glycogen. Glycogen breaks down into a substance called pyruvate and, in the process, produces energy. We often refer to this process as anaerobic energy because it does not use any oxygen. When pyruvate breaks down further, it produces much more energy. This energy is called aerobic because this process uses oxygen. If pyruvate does not break down, it usually turns into lactate. When pyruvate is produced, the muscle cell will try to use it for aerobic energy. However, if the cell does not have the capacity to use all the pyruvate produced, it will be changed chemically to lactate. Some cells have a large capacity to use pyruvate for aerobic energy while others have very little. With training, many cells can adapt to use more pyruvate and, thus, produce less lactate. Lactate is present in our system at rest and as we go about our every day activities, although at low levels. However, as exercise or work activity increases in intensity, large amounts of pyruvate are produced very quickly. Because pyruvate can be produced quickly, not all of it may be used for aerobic energy. The surplus pyruvate will turn into lactate. This is why lactate is such a significant marker for training. When it is produced, it is a sign that aerobic energy is limited during the activity. There is a different reason why more lactate is produced as exercise intensity increases. As exercise increases, extra muscle fibers will be recruited. These fibers are used infrequently at rest or in light activity. Fast twitch fibers are not very good at turning pyruvate into aerobic energy. Hence, a lot of this pyruvate turns into lactate. 2. What is the metabolic fate of blood lactate in normal subjects Lactate is a major metabolic intermediate. Its fate depends on the conditions of the cell. In aerobic oxidation of glucose, glucose is converted to pyruvate, which is then converted to acetylSCoA (or AcetylCoA), a high-energy thioester. Under anaerobic conditions, pyruvate is reduced to lactate to regenerate the NAD + needed in Step 6 of glycolysis to keep glycolysis pathway going. 3. Which enzyme is responsible for lactate production Write out the equation for this reaction. Lactate Dehydrogenase (LDH) is an enzyme responsible for lactate production, which is present in varieties or organisms including plants and animals. This will catalyse the inter-conversion of pyruvate and lactate with concomitant inter-conversion of NADH and NAD+. As it can also catalyze the oxidation of hydroxybutyrate, occasionally called Hydroxybutyrate Dehydrogenase (HBD). Or D(-)Lactate + -NAD + Hydrazine LDH> Pyruvate Hydrazone + -NADH Abbreviations used: -NAD = -Nicotinamide Adenine Dinucleotide, Oxidized Form -NADH = -Nicotinamide Adenine Dinucleotide, Reduced Form LDH = D-Lactic Dehydrogenase 4. The student decided to use this enzyme to measure blood lactate levels. He reasoned that if he could produce reaction conditions that allowed the enzyme to metabolise all the lactate present, he would be able to calculate the lactate content of his sample by reading the change in absorbance at 340nm. Explain the reasoning behind this idea. LDH catalyzes the reduction of pyruvate to L-lactate with concomitant oxidation of NADH2 to NAD. Since the oxidation of NADH2 is directly proportional to the reduction of pyruvate in equimolar amounts, the LDH activity can be calculated from the rate of decrease in absorbance at 340 nm (334 nm or 365 nm). 5. In order to obtain suitable conditions for an assay to measure plasma lactate levels, the student chose a buffer containing 1.5% (w/v) hydrazine. Hydrazine reacts covalently with pyruvate to form a hydrazone: CH3 CH3 | | C=O + NH2.NH2 C=N.NH2+H20 | | COOH COOH What effect will hydrazine have on this reaction How will the addition of hydrazine to the reaction mixture help to measure lactate levels, assuming that all other reagents are present in excess The hydrazine destroys the pyruvate, allowing the reaction to run to the complete oxidation of all lactate molecules. To ensure this, NAD+ is provided in excess. The concentration of lactate in the sample is proportional to the increase in absorbance as NAD+ is reduced to NADH. 6. The student then set up an experiment to produce a calibration graph for absorbance at 340nm against (lactate), as shown below. He also tested two dilutions of a plasma sample from a patient. He used a stock solution of 2.5mM lactate, and kept the total volume in each cuvette constant at 4.0 ml. He incubated the samples with enzyme for 30 minutes and then recorded the change in absorbance at 340nm. He checked the absorbance values again after a further 5 minutes; they had not changed. Why was it necessary to check the absorbance values twice It was necessary to check the absorbance values twice to be sure of the stability of the solution, i.e. to ensure that all the lactate molecules have been broken down and the solution has reached stability. 7. Calculate the lactate concentration in each tube, showing your working clearly. Complete the results table and plot the calibration graph. Remember to present your graph to a high standard. The corrected Absorbance change would be determined by subtracting the value derived from the 0 lactate control from all sample readings. The 0 lactate control gave Absorbance of 0.1 (340nm). Subtracting 0.1 from the Absorbance values for the reading, the corrected absorbance is as reflected in the table below: Tube Vol. stock lactate added (ml) (lactate) in tube (Um) Abs change at 340nm Average absorbance at 340nm Corrected absorbance at 340nm 1 0 0 0.01 2 0 0 0.01 3 0.02 0.02 0.10 0.09 4 0.02 0.02 0.08 0.07 5 0.04 0.04 0.160 0.15 6 0.04 0.04 0.171 0.161 7 0.06 0.06 0.248 0.238 8 0.06 0.06 0.240 0.230 9 0.08 0.08 0.329 0.319 10 0.08 0.08 0.321 0.311 11 0.10 0.10 0.389 0.379 12 0.10 0.10 0.410 0.40 15 Patient sample 0.1ml 214% of 0.1ml = 0.214mg/dl 0.731 0.721 16 Patient sample 0.1ml 214% of 0.1ml = 0.214mg/dl 0.751 0.741 0.741 17 Patient sample 0.05ml 214% of 0.05 = 0.107mg/dl 0.35 0.25 18 Patient sample 0.05ml 214% of 0.05 = 0.107 0.42 0.385 0.32 With control stock lactate of 0.1ml Corrected Absorbance = 0.311 and 0.379 Mean Absorbance for 0.1ml of control stock lactate = 0.311+ 0.379/2 = 0.345 With 0.1ml of Patient Sample Corrected Absorbance = Mean Absorbance for 0.1ml Patient Sample = 0.721 + 0.741/2 = 0.731 Thus the % difference = 0.731 X 100 = 214% 0.379 1 i.e. the lactate concentration of the patient sample is 214% of the control stock used for the same volume. 8. Determine the lactate concentration in the original patient plasma sample. Explain your working. If 0.1ml of the sample have a lactate concentration of 0.214mg/dl 1.0ml of sample will contain: 0.214mg/dl x 10 = 2.14 Thus: 4.0ml (the total plasma sample) will contain: 2.14 x 4 = 8.56mg/dl 9. The normal range for plasma (lactate) is 0.5-2.0mM lactate. Research possible causes for a change in plasma lactate levels. Comment on, and suggest explanations for the result obtained for this patient. (500 word max) Under fluctuating environmental conditions, living organisms, especially man, have developed, through the processes of adaptation and evolution, their own characteristic patterns of physiological compensation mechanisms for maintaining internal physiological homeostasis at the expense of energy. With changes in external environments, physiological homeostasis is achieved through the process of capacity adaptation over a normally varying environment, or that of resistance adaptation at environmental extremes. In the process, the most efficient source of energy is derived from the transformation of adenosine phosphate compounds under aerobic conditions. However, organisms are also capable of utilizing alternative energy sources, especially under the acute stimulation of environment stress or burst movements. The common result of incomplete tissue oxygen supply, which is a characteristics of excess workload, such as stress (under healthy conditions), is the breakdown of creatine phosphate and ATP, and the production of intermediary glycolytic metabolic end products, with the most prominent one in vertebrates being lactate. Accordingly, blood concentration of lactate could be a biological indicator for stress in patients. Moreover, in diseased condition that result in tissue hypoxia, excessive lactate acid could be present in the plasma either because of increased aerobic production or because lactate acid clearance from the blood is impaired. Therefore, in the patient under study, the high amount of lactate in the patient's blood (8.56mg/dl) could either be as a result of increase stress following the temporary anxiety of taking blood sample or due to tissue hypoxia caused by an underlying disease. Read More