Measurement of Lactate Dehydrogenase Isoenzymes - Essay Example

Measurement of LDH isoenzymes affiliation" Cellular signaling is a fascinating process,where protein receptors, transducers, accelerators, inhibitors, and many other substances interact to produce a cellular response. Enzymes play a critical role in cellular functioning, and a single enzyme deficiency may cause our cells to fail. With a tremendous advance in medical and laboratory analysis during the past years, researchers have been able to find information from the measurement of enzymes. In most cases, a clinical diagnosis, based on signs and symptoms, is complemented by laboratory tests, which bring answers to clinical questions and decisions. 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. Because these different isoforms are found in distinct locations, its measurement is key when establishing the site of damage. 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 2-oxobutyrate, and compared to the former, as a possible means of predicting LDH isoenzyme profiles. 1. How do the spectra of NAD+ and NADH differ 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 (2008) there are normally five isoenzymes of LDH (LDH1-5) expressed in living cells, made of the combination between M-polypeptide and H-polypeptide. M chains catalyze the conversion of pyruvate to lactate, whereas H chains improve the aerobic oxidation of pyruvate. As a result, an increase in the number of M chains makes that isoenzyme more prone to the anaerobic pathway. LDH5 is composed of four M monomers, LDH4 is composed of three M monomers and one H monomer, and so on (Koukourakis, 2003). LDH is expressed in all living cells, with a predominance of LDH1 because, normally, aerobic oxidation is the primary cellular mechanism. It is important to know the normal location of the different isoenzymes; LDH1 and LDH2 are both located in the heart and red blood cells; LDH3 is found in the lung tissue; LDH4 and LDH5 are located in the striated (skeletal) muscle and liver. Normal ratios are LDH1 < LDH2, and LDH5 < LDH4. By using laboratory analysis, measurements of these different isoenzymes help in the differential diagnosis. For instance, it is known that a ratio LDH1 >LDH2 is found in conditions such as myocardial infarction, hemolytic anemias, pernicious anemia, folate deficiency, and renal infarct. An abnormal ratio LDH5 >LDH4 is seen in cirrhosis, hepatitis, and hepatic congestion (Ferri, 2009). 3. What is the significance of these results to using LDH to diagnose disease (or assess toxicity) LDH may be elevated in several conditions, such as Infarction of myocardium, lung, and kidney; diseases of the cardiopulmonary system, liver, collagen, and central nervous system; as mentioned above, it may also increase in hemolytic and megaloblastic anemias, but also in transfusions, seizures, muscle trauma, muscular dystrophy, acute pancreatitis, hypotension, shock, infectious mononucleosis, inflammation, neoplasia, intestinal obstruction, and hypothyroidism. Total levels of the enzymes depend on age, gender, race, muscle mass, physical activity, and weather condition (Brancaccio, 2008). 4. Increased release from damaged or diseased cells is probably the major mechanism by which enzyme activity in blood is increased. There are at least three other mechanisms by which enzyme activities can be increased in plasma during disease or toxicity. What are these additional mechanisms As we have seen, measurement of plasma enzyme activities is of value in diagnostic tests. According to Turecky (2004), "blood plasma enzymes can be divided into primarily intracellular enzymes", being released when there is damage of cell membranes", and "enzymes which are actively secreted into the blood, where they fulfill their physiological function". The value of enzymes as disease markers relies on adequate measurement and biochemistry knowledge when interpreting results. Additionally, it is important to correlate the medical history with laboratory results. In normal subjects, levels reflect the balance between the rate of synthesis and release into plasma. Besides enzyme release from damaged cells, Turecky mentions other mechanisms by which enzyme activity can be increased. These include "proliferation of cells, and increase in the rate of cell turnover, or reduced clearance". An induction in the rate of enzyme synthesis, and the formation of macroenzymes, which result from binding of regular enzymes to another high molecular weigh plasma component, are other involved mechanisms. It can be inferred that increased enzyme levels in the serum may be the result of increased formation from a malignant tumor, or decreased elimination as a result of obstruction of excretory pathways. 5. Enzymes are best measured for diagnostic purposes in serum rather than plasma. Why (What is the difference between plasma and serum). Although plasma is more easily separated from the cellular constituents, certain enzymes are measured in serum because of method dependent differences, found in several studies. According to Miles et al. (2004), Significant differences between serum and heparinized plasma results have been reported for albumin, alkaline phosphatase, calcium, carbon dioxide, chloride, creatine kinase, glucose, lactate dehydrogenase, inorganic phosphorus, potassium, and total protein. The study conducted by the researchers showed that serum and plasma samples give results that differ enough. For potassium, both serum and plasma reference intervals are readily available, and plasma is the preferred sample type. For the other assays, they do recommend that only serum samples be accepted. For some enzyme measurements, plasma is preferable to serum because the time between collection and analysis is shortened, since anticoagulated blood can be centrifugated immediately. "The use of plasma eliminates clogging of probes by fibrin strings, which are frequently formed in serum" (Doumas et al., 1989). Serum LDH is, on average, 30 IU/L higher than plasma LDH, owing to release of LD from platelets (McPherson, 2007). 6. When measuring serum LDH as an indicator of disease/toxicity, it is imperative to avoid haemolysis, why Given that total LDH is expressed in all living cells, when drawing blood and sending for analysis, haemolysed samples should be avoided because red blood cells contain high levels of LDH. The samples must be centrifuged and cells separated from serum. Moreover, LDH has long been considered a clinical marker of intravascular hemolysis; its serum levels are substantially elevated with conditions where intravascular hemolysis is a major component. 7. As well as LDH, isoenzymes also exist for creatine kinase (CK) and acid phosphatase (ACP). Are these isoenzymes of any clinical or diagnostic use If so, describe briefly how they could be measured in a hospital pathology laboratory. Monitoring of CK and characterization of its isoenzymes is widely used in the diagnosis of myopathies, cardiomyopathies, and encephalopathies. CK-MB is a reliable marker of myocardial necrosis. In crush syndrome, CK levels are used as a prognostic measurement. Patients who have neurological conditions, such as acute cerebrovascular accidents, show marked elevation of CK-BB. Primary skeletal muscle disorder manifests with various symptoms and serum CK elevation (Brancaccio, 2008). Total acid phosphatase (ACP) is measured by its ability to cleave phosphate groups at an acid pH. Some of the substrates and conditions used to measure enzymatic activity with increased specificity are thymolphthalein monophosphate and alpha-naphthyl phosphate. Isoenzymes of ACP can be separated by electrophoresis, for prostate and bone studies. Immunoassays for both prostatic and bone isoenzymes have been developed (McPherson, 2007). In conclusion, Lactate dehydrogenase (LDH) is an important enzyme, which is only used in anaerobic processes. By converting pyruvate to lactate, it reoxidizes NADH to NAD. There are normally five isoenzymes of LDH in living cells, made of different polypeptide combinations. It is important to know the normal location of the different isoenzymes, as their value as disease markers relies on adequate measurement and biochemistry knowledge when interpreting results. Serum LDH is higher than plasma LDH, and its measurement is best done in serum. Hemolysis must be avoided during the procedure, because red blood cells contain high levels of LDH. References Brancaccio, P. (2008). medicine. Clinical Sports Medicine, 27, 1-18. Doumas, B. House, L. et al. (1989). Differences between values for plasma and serum in tests performed in the Ektachem 700 XR analyzer, and evaluation of "plasma separator tubes (PST)". Clinical Chemistry, 35, 151-153. Ferri, F. (2009). Ferri's Clinical Advisor 2009 (1st ed.). Mosby, Philadelphia. Koukourakis, M.I.Giatromanolaki, A.Sivridis, E. (2003). Lactate dehydrogenase isoenzymes 1 and 5: differential expression by neoplastic and stromal cells in non-small cell lung cancer and other epitelial malignant tumors. Tumor Biology,24, 199-202. McPherson, R. Pincus, M. (2007). Henry's Clinical Diagnosis and Management by Laboratory Methods. (21st ed.). Elsevier Saunders Company. Miles, R. Roberts, R. Putman, A. et al. (2004). Comparison of Serum and Heparinized Plasma Samples for Measurement of Chemistry Analytes. Clinical Chemistry, 50, 1704-1706. Turecky, L. (2004). Macroenzymes and their clinical significance. Bratisl Lek Listy, 105, 260-263. Read More