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.
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).
D(-)Lactate + -NAD + Hydrazine LDH> Pyruvate Hydrazone + -NADH
-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:
C=O + NH2.NH2 C=N.NH2+H20