Lactate + NAD+ rightarrow pyruvate + NADH is catalysed by lactate dehydrogenate
ID: 65767 • Letter: L
Question
Lactate + NAD+ rightarrow pyruvate + NADH is catalysed by lactate dehydrogenate (LD) The UV spectra (using 1cm cuvettes) of 20 mu M solutions of lactate, pyruvate, NAD+ and NADH are shown below In Fig 1 what is the e_340 of NADH? What is the e_280 of NAD+. A mixture of lactate and NAD+ were placed In a 1cm cuvette and lactate dehydrogenase (LD) solution added at time t = 0, Fig2 shows the plot of Absorbance at 340nm with time Calculate the rate of this reaction in mu moles/L/min. Why specifically was 340nm used in this assay? Why specifically was 340nm used in this assay? (2 reasons) The above represents how LD 'activity' levels are determined in hospital clinical labs. Find out why serum levels of LD are used diagnostically for what specific diseases or conditions Would elevated or decreased LD activity levels be found in patients with the specific diseases or conditions you have described above. Explain you answer. 5 isoenzymes/isoforms of LD actually exist in humans. What are isoenzymes? Find out how isoenzymes of LD analysis are used diagnostically.Explanation / Answer
1. From the Fig 1 the e340 of NADH is about 5.8 absorbance units and e280 of NAD+ is about 3.4 absorbance units.
You can calculate this Vo value by taking the difference between any two absorbance values within the linear portion of the curve and then dividing by the difference in time between these values.
Vo= A340 (time 2) - A340 (time 1)/time 2 (seconds) - time 1 (seconds)
Vo= A340 second
Then convert the initial velocity for each reaction to a change () in the absorbance at 340 nm per minute (A340/minute) as follows:
Vo= A340 x 60 seconds second minute
= A340 per minute
2. It is more convenient to assay LDH activity in the direction of L-lactate oxidation because NADH absorbs light at 340 nm and NAD+ does not. The reaction thus can be measured as an increase in A340.
3. A lactate dehydrogenase (LD) test is a non-specific test that may be used in the evaluation of a number of diseases and conditions. LD is an enzyme that is found in almost all of the body's cells (as well as in bacteria) and is released from cells into the fluid portion of blood (serum or plasma) when cells are damaged or destroyed. Thus, the blood level of LD is a general indicator of tissue and cellular damage.
4. High LDH Levels
High levels of LDH indicate some form of tissue damage. High levels of more than one isoenzyme may indicate more than one cause of tissue damage. High levels of all five LDH isoenzymes could indicate multiple organ failure.
Low LDH Levels
LDH deficiency affects how the body breaks down sugar for use as energy in cells, particularly muscle cells. It’s very rare for a person to have low LDH levels.
5. There are five different forms of LDH, and they are distinguished by slight differences in their structure. Each form of the LDH enzyme is called an isoenzyme. The isoenzymes of LDH are LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5.
Different LDH isoenzymes are found in different body tissues. The areas of highest concentration for each type of isoenzyme are:
6. Because LDH is in so many tissues throughout the body, complete LDH levels alone won’t be enough to determine the location and cause of your tissue damage. A diagnosis will also require measuring the levels of LDH isoenzymes. For example, high LDH-4 and LDH-5 may mean either liver damage or muscle damage, but liver disease can’t be confirmed without a full liver panel.
It’s normal for a person to have a higher level of LDH-2 than LDH-1. After a heart attack, however, the level of LDH-1 rises and is usually higher than the level of LDH-2. This is called a flipped pattern. Total LDH level will rise within 24 to 72 hours after a heart attack and peak in two to four days. It will return to normal in about 10 to 14 days. A test for troponin, a protein in myocardial cells, is a more accurate indicator of a heart attack.
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