LDH isoenzymes

SCH 28191 (experimental drug) Probe 6. beta-carotene Probe 7. LDH isoenzymes Probe 8. HGH tryptic digest Probe 9. urea, thiourea Probe 10. tricyclic antidepressants Probe 11. avermectins... 

Elevated pyruvate concentration inhibits the heart muscle lactate dehydrogenase (LDH) isoenzyme but not the skeletal muscle LDH isoenzyme. 

An example of an enzyme which has different isoenzyme forms is lactate dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of the coenzyme NADH (see above). LDH is a tetramer of two different types of subunits, called H and M, which have small differences in amino acid sequence. The two subunits can combine randomly with each other, forming five isoenzymes that have the compositions H4, H3M, H2M2, HM3 and M4. The five isoenzymes can be resolved electrophoretically (see Topic B8). M subunits predominate in skeletal muscle and liver, whereas H subunits predominate in the heart. H4 and H3M isoenzymes are found predominantly in the heart and red blood cells H2M2 is found predominantly in the brain and kidney while HM3 and M4 are found predominantly in the liver and skeletal muscle. Thus, the isoenzyme pattern is characteristic of a particular tissue, a factor which is of immense diagnostic importance in medicine. Myocardial infarction, infectious hepatitis and muscle diseases involve cell death of the affected tissue, with release of the cell contents into the blood. As LDH is a soluble, cytosolic protein it is readily released in these conditions. Under normal circumstances there is little LDH in the blood. Therefore the pattern of LDH isoenzymes in the blood is indicative of the tissue that released the isoenzymes and so can be used to diagnose a condition, such as a myocardial infarction, and to monitor the progress of treatment. 

LDH. Rather surprisingly, the properties of LDH have been investigated fully only in Hymenolepis spp. Burke et al. (108) purified 128-fold a single species of LDH from H. diminuta which resembled the H form of mammalian LDH. Other workers (448, 567, 630, 926) were able to distinguish electrophoietically at least two LDH isoenzymes in H. diminuta and H. microstoma. The kinetic parameters for LDH, similar in both species (Table 5.7), indicate that pyruvate reduction is favoured over lactate oxidation and that pyruvate, once formed, would be rapidly reduced to lactate, with the subsequent oxidation of NADH. 

Several markers should no longer be used to evaluate cardiac disease, including aspartate aminotransferase, total CK, total lactate dehydrogenase (LDH), and LDH isoenzymes. Due to their wide tissue distribution, these markers have poor specificity for the detection of cardiac injury. Because total CK and CK-MB have served as standards for so many years, some laboratories may continue to measure them to allow for comparisons to cardiac troponin over time, before discontinuing use of CK and CK-MB. In addition, the use of total CK in developing countries may be the preferred or only alternative for financial reasons. However, it should be clear that, for monitoring ACS patients to assist in clinical classification, cardiac troponin is the preferred biomarker. 

Total LDH activity was significantly enhanced in EDL, diaphragm, and serum by carbofuran (1.5mg/kg, s.c.) or methyl parathion (5 mg/kg, i.p.) within 1 h of injection. Each AChE inhibitor caused marked elevation of all five isoenzymes in serum, with maximum increases in LDH-1 and LDH-4 (three-fold). Unlike serum, muscle LDH isoenzymes depicted variable patterns by carbofuran or methyl parathion intoxication. A significant decline in ATP appears to be the mechanism involved in leakage of cytoplasmic/mitochondrial enzymes into circulation (Gupta et al, 1994). 

Using a method based on the oxamate-agarose column of O Cara and Barry, it has been found possible to separate the LDH-X isoenzyme from the other LDH isoenzyme in mouse testes [76]. Unlike the other isoenzymes, LDH-X had no affinity for the column. 

While each of the five LDH isoenzymes catalyzes the conversion of lactic acid to pyruvic acid, the isoenzymes are produced in different organs. Because of this, the polypeptide moieties and the rates at which lactate can be converted to pyruvate are slightly different for each isoenzyme. Similarly, different species often possess identical metabolic pathways, and have equivalent but slightly different enzymes that catalyze identical reactions. The differences that occur within such a family of enzymes usually occur in noncritical regions of the polypeptide moiety, by the substitution of one amino acid residue for another, or by the deletion of amino acid residues. 

Figure 14.8. Bioelution of LDH isoenzymes from a Sepharose AMP affinity column using a concave NADH gradient. [Reprinted, by permission, from P. Brodelius and K. Mosbach, FEBS Lett. 35, 1973, 223-226. Separation of the Isoenzymes of Lactate Dehydrogenase by Affinity Chromatography Using an Immobilized AMP-Analogue . The exclusive for all languages and countries is vested in the Federation of European Biochemical Societies.]...

Isoenzymes. Weber (W3), Grosfield et al. (G15, G16), and Marghescu (M5) have all examined the LDH isoenzyme patterns in normal and psoriatic skin. The pattern in both is that of an anaerobic tissue, i.e., a predominance of the cathodal bands 4 and 5. Psoriatic lesions tend to have increased activity and a slight shift to a more aerobic pattern. 

As first pointed out by Dreyfus et al. (Dll), the abnormal LDH isoenzyme pattern in diseased muscle resembles that of normal fetal muscle. In fact, in dystrophic chickens the normal adult isoenzyme pattern is never attained in the affected birds (D3), and possibly this is true of some human muscle diseases. An increase in the specific activity of many other glycolytic enzymes during development has been reported (B5, K3). 

LDH (M.W. 134,000) oceurs as five tetrameric isoenzymes composed of two different types of subunits. Subunits M (for muscle) and H (for heart) are encoded by loci in chromosomes 11 and 12, respectively. Two subunits used in the formation of a tetramer yield five combinations H4(LDH-1), H3M(LDH-2), H2M2(LDH-3), HM3(LDH-4), and M4(LDH-5). The tissue distributiont of LDH isoenzymes is variable. For example, LDH-1 and LDH-2 are the principal isoenzymes in heart, kidney, brain, and erythrocytes LDH-3 and LDH-4 predominate in endocrine glands (e.g., thyroid, adrenal, pancreas), lymph nodes, thymus, spleen, leukocytes, platelets, and nongravid uterine muscle and LDH-4 and LDH-5 preponderate in liver and skeletal muscle. In tissue injury or insult, the appropriate tissue isoenzymes appear in plasma (Chapter 8) thus, determination of LDH isoenzyme composition has diagnostic value. 

Serum LDH isoenzymes can be separated by electrophoresis on agarose gel or cellulose acetate membrane, usually at pH 8.6. After separation, their location is determined by incubation of the support medium in a... 

Fluorescence in region at anodic band corresponding to albumin NADH produced by substrate overlay in fluorometric scan of LDH isoenzymes... 

Figure E38-1. LDH isoenzyme patterns for several human diseases. (Courtesy - Gelman Instrument Co., Ann Arbor, Ml)...

Visual localization of electrophoretically separated LDH isoenzymes is accomplished by the reduction of nitro-tetrazolium blue as the electron acceptor (terminal) in a medium containing phenazine methosulfate and NAD. The linked reaction is as follows ... 

You obtain about 10 mL of blood from his horse, take it back to the lab, and perform an LDH isoenzyme separation, (heparin not needed, wear... 

Adding the overlay. There are certainly more than 5 compounds on that strip. What is needed is to locate the 5 LDH isoenzymes. This is done by using a selective dye and the technique is called hstochemistry. The result is a zymogram. With a pipet, spread 1-2 mL of the solution in the low actinic flask over the top of the gel. This is called an overlay. 

Clean out the syringes by pumping water through them. the LDH isoenzymes... 

A profile of the serum isoenzymes of lactate dehydrogenase, (a) The pattern of LDH isoenzymes from a normal individual, (b) The pattern of LDH isoenzymes of an individual suffering from a myocardial infarction. 

Muscle is the only source of CK and by measuring the isoenzyme CK-MB one can determine whether or not cardiac muscle is involved. If the liver is involved then the serum yGT should be increased as this is one of the most sensitive indicators of liver disease. LDH isoenzyme analysis will help identify erythrocyte damage as a possible source for some of the LDH and AST activity. In haemolytic disorders, one would expect a reticulocytosis and intravascular haemolysis will lead to a low serum haptoglobin level. These investigations will help identify whether or not erythrocytes have contributed to the serum enzymes. 

Uke the other enzymes used in thediagnosisofMI.l.DH is not specific to cardiac muscle, being found in liver and red cells. Although there is a measurable difference between the LDH, isoenzyme in heart and the LDH, isoenzyme in liver, the red cells can al.so release LDH,. Blood for LDH measurement must therefore not be allowed to remain unseparated before analysis, or in vitro haemolysis w ill invalidate the LDH measurements. 

Lactate produced by smooth muscle of swine carotid artery has been shown to be quantitatively derived from extracellular glucose, whereas gly-cogenolysis provides carbohydrates for oxidative metabolism (Lynch and Paul, 1983, 1987). Interestingly, the lactate dehydrogenase (LDH) isoenzyme pattern varies between slow (aorta) and fast (portal vein and bladder) smooth muscle in rat, such that aorta has a higher proportion of the H subunit, which has a higher affinity for lactate and is product-inhibited at lower concentrations of lactate (Malmqvistef flZ., 1991). Thus the slow aortic muscle is more adapted for oxidative metabolism than the faster bladder and portal vein muscles. 

See also LDH Isoenzymes, Pyruvate/Lactate/Ethanol Metabolism, Anaerobic Process for Generating Metabolic Energy, Lactic Acid Fermentation, Ethanol Metabolism and Gluconeogenesis... 

The tissue specificity of isoenzyme patterns of LDH is useful in clinical medicine. Such pathological conditions as myocardial infarction, infectious hepatitis, and muscle diseases involve cell death of affected tissue, with release of cell contents to the blood. The pattern of LDH isoenzymes in the blood serum is representative of the tissue that released the isoenzymes. This information can be used to diagnose such conditions and to monitor the progress of treatment. 

HHMM, and HMMM, respectively (Markert, Shaklee, and Whitt 1975). The widespread tissue distribution of LDH differs with the various species (Garbus et al. 1967 Cornish, Barth, and Dodson 1970 Karlsson and Larsson 1971 Schultze et al. 1994). Some additional isoenzymes of LDH have been described, and some LDH isoenzymes may complex with drugs—for example, streptokinase (Podlasek and McPherson 1989). Because LD is a cytoplasmic enzyme, it is increased in serum during hepatocyte or myocyte necrosis and conditions that are toxic to hepatocytes or myocytes. However, it lacks specificity and sensitivity when compared to other enzymes for detection of specific organ injury. In laboratory animals, the primary use for LD has been the detection of experimentally induced myocardial injury (see Chapter 7). 

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