Substances levels can give a lot of information to healthcare professionals, in both clinical and research settings.
Lactate dehydrogenase (LDH) is present in many human tissues, enabling cells to undergo anaerobic glycolysis; its main cellular function, conversion of pyruvate into lactate, provides the energy production cycle with more substrates. Normal and abnormal levels are now standardized, and measuring its activity in serum/plasma will help elucidate the origins of damage or disease. There are different isoenzymes of LDH, which differ in their structure and certain other properties.
In this practical work, which is divided into 3 weeks, we will first make a comparison of the absorption spectra of NAD+ and NADH, since the assay for LDH makes use of an important difference in these spectra. In addition, the linearity of the LDH assay, with respect to the amount of enzyme, will be assessed, and the limit of linearity determined. In week two, we will assess the LDH isoenzyme profile in rat serum and selected tissues, using agarose gel electrophoresis. Finally, the total LDH activity will be determined, using its natural substrate pyruvate. LDH activity will also be measured using the substrate analogue
Nicotinamide adenine dinucleotide (NAD) is a coenzyme, a molecule which aids an enzyme in the acceleration of a chemical reaction, or catalysis. NAD is an agent that accepts electrons from other substrates; when NAD is reduced, the reaction forms NADH, a molecule that can be used as a donator of electrons. In aerobic glycolysis, for instance, energy is produced in the form of 2 adenosine triphosphates (ATP); in order to produce the adequate amount of energy the body cells need, the cellular mitochondria utilizes NADH. The inner membranes of this organelle are impermeable to NADH, so the latter is re-oxidized to NAD and delivers its electrons to certain shuttles that are able to transfer the substrate into the mitochondria.
By these means, cytoplasmic NADH is oxidized and yields 3 ATP molecules, much more energy to the cell. Many other enzymes produce NADH in the mitochondria, all of which can be oxidized in the electron transport chain and in the process, capture energy for ATP synthesis. Once the NADH has been oxidized, the NAD can again be used by enzymes that require it, including those of the citric acid cycle (Krebs's cycle), and pyruvate dehydrogenase, among others.
2. Compare and comment on the distribution of the LDH isoenzyme bands within the tissues.
Glycolysis is a biochemical cascade that coverts the main body fuel, glucose, into two pyruvates, releasing energy. In certain cells, where oxygen lacks, glycolysis occurs anaerobically: red blood cells, skeletal muscle, etc. Lactate dehydrogenase (LDH) is an enzyme that is only used in anaerobic processes. By converting pyruvate to lactate, it reoxidizes NADH to NAD, so that a new reaction can be started using this coenzyme. This is an important function of LDH, because energy production would stop without NAD substrates.
According to Brancaccio