Isoenzyme concentration

Principle (immunoinhibition method) Antihuman CK-M antibodies contained in the slide inhibits all CK-MM activities and approx. 50% of the CK-MB activity during the lag phase of approx. 3.5 minutes. The remaining CK activity represents 50% of the total CK-MB isoenzyme concentration plus any CK-BB. 

This reaction provides ATP for muscle contraction (Chapter 21). Skeletal muscle contains predominantly CK3, whereas heart muscle (myocardium) contains CK3 and CK2. Serum normally contains a small amount of CK3 derived predominantly from skeletal muscle. Detection of CK2 in serum (in an appropriate clinical setting) is strongly suggestive of myocardial damage. Since an abnormal isoenzyme level may occur with apparently normal total activity of the enzyme, determination of the isoenzyme concentration is essential in the diagnostic enzymology. 

D. Neumeier, Measurement of Creatine Kinase Isoenzyme Concentration by Immunoassay, in Creatine Kinase Isoenzymes Pathophysiology and Clinical Application (ed. H. Lang), Springer-Verlag, New York, 1981, p. 75. 

D7. Di llio, C., Del Boccio, G., Aceto, A., and Federici, G., Alteration of glutathione transferase isoenzyme concentrations in human renal carcinoma. Carcinogenesis (London) 8, 861-864 (1987). 

Variations between species using different enzyme substrates have been shown for cholinesterase (Myers 1953 Evans 1990) and other enzymes (e.g., angiotensin converting enzyme Evans 1989). For alkaline phosphatase, the majority of methods employ 4-nitrophenylphosphate as the substrate, but there are two main alternative buffers—diethanolamine and 2-aminopropanol—that can cause interspecies differences (Masson and Holmgren 1992). For isoenzymes, the majority of laboratories currently use a variety of electrophoretic separation methods although selective inhibition of isoenzymes with antibodies is being used increasingly, there are problems associated with protein specificity and relative isoenzyme concentrations in animal samples. 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

Antithyroid drags have several side effects. The most frequent side effects are maculopapular rashes, pruritus, urticaria, fever, arthralgia and swelling of the joints. They occur in 1-5% of patients [1, 2]. Loss of scalp hair, gastrointestinal problems, elevations of bone isoenzyme of alkaline phosphatase and abnormalities of taste and smell are less common. The incidence of all these untoward reactions is similar with MMI and PTU. Side effects of MMI are dose-related, whereas those of PTU are less clearly related to dose [1]. PTU may cause slight transient increases of serum aminotransferase and y-glutamyl transpeptidase concentrations but also severe hq atotoxicity whereas methimazole or carbimazole can be associated with cholestasis. The side... 

Imidazole antimycotics, ketoconazole, clotrimazole, and miconazole are potent inhibitors of various cytochrome P450-isoenzymes that also affect the metabolism of retinoids. They were fust shown to inhibit the metabolism of RA in F9 embryonal carcinoma cells. When tested in vitm liarazole, a potent CYP-inhibitor, suppressed neoplastic transformation and upregulated gap junctional communication in murine and human fibroblasts, which appeared to be due to the presence of retinoids in the serum component of the cell culture medium. Furthermore, liarazole magnified the cancer chemopreventive activity of RA and (3-carotene in these experiments by inhibiting RA-catabolism as demonstrated by absence of a decrease in RA-levels in the culture medium in the presence of liarazole over 48 h, whereas without liarazole 99% of RA was catabolized. In vivo, treatment with liarazole and ketoconazole reduced the accelerated catabolism of retinoids and increased the mean plasma all-irans-RA-concentration in patients with acute promyelocytic leukemia and other cancels. 

In resting muscle the high concentration of ADP does not decrease the proton gradient effectively and the high membrane potential slows electron transport. ADP, formed when ATP is hydrolyzed by myosin ATPase during contraction, may stimulate electron transport. However, the concentration of ATP (largely as its Mg salt) is buffered by its readily reversible formation from creatine phosphate catalyzed in the intermembrane space, and in other cell compartments, by the various isoenzymes of creatine kinase (reviewed by Walliman et al., 1992). 

The uptake of glucose into the liver is independent of insulin, but liver has an isoenzyme of hexokinase (glucokinase) with a high K, so that as the concentration of glucose entering the liver increases, so does the... 

HT is metabolised primarily by MAO to 5-hydroxyindoleacetic acid (5-HIAA) (Fig. 9.4). In vitro, 5-HT is the preferred substrate for the MAOa, rather than the MAOb isoenzyme (see Chapter 8) and this appears to be the case in vivo since MAOa, but not MAOb, knock-out mice have increased concentrations of 5-HT in the brain. Obviously, because of its indole nucleus, 5-HT is not a substrate for the enzyme COMT which metabolises the catechol derivatives, dopamine and noradrenaline. However, other metabolic products of 5-HT are theoretically possible and one, 5-hydroxytryptophol,... 

In addition to all of the expected enzyme systems present in leaf tissue, fresh tea leaves contain a high level of polyphenol oxidase that catalyzes the oxidation of the catechins by atmospheric oxygen. Tea polyphenol oxidase exists as series of copper-containing (0.32%) isoenzymes. The major component has a molecular weight of about 144,000.54 The enzyme is concentrated in the leaf epidermis.55 Soil copper deficiency is sometimes responsible for inadequate oxidation during processing.56... 

By using in vitro preparations of human enzymes it is possible to predict those antibiotics that will adversely affect the metabolism of other drugs [110]. Such studies have shown that rifaximin, at concentrations ranging from 2 to 200 ng/ml, did not inhibit human hepatic cytochrome P450 isoenzymes 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 [34], In an in vitro hepatocyte induction model, rifaximin was shown to induce cytochrome P450 3A4 (CYP3A4) [34], an isoenzyme which rifampicin is known to induce [109],... 

The clinical picture includes cramps and recurrent myoglobinuria following intense exercise. Aside from episodes of myoglobinuria, none of the patients was weak. Forearm ischemic exercise caused a 1.5-2.0-fold increase in venous lactate concentration, an abnormally low but not absent response. Muscle biopsy showed normal or only moderately increased glycogen concentration. Because other accessible tissues, such as erythrocytes, leukocytes and cultured fibroblasts, express a different isoenzyme, the diagnosis of PGM-M subunit deficiency must be established by biochemical studies of muscle. Four different... 

The H-rich forms found in heart and red blood cells tend to operate at low lactate concentration, whilst the M-rich forms work better when lactate concentration is high. Further details about LD isoenzymes are given in Chapters 6 and 7. 

The third and final control step is mediated by PK. This enzyme, like HK, exists as a number of isoenzymes in different tissues and, like the PFK reaction, is controlled by both the concentration of metabolites and covalent effects. Furthermore, PK also illustrates two other means of metabolic control, namely enzyme induction and feedforward, regulation. 

LD occurs in various tissues of the body as five isoenzymes details are given in T able 6.7. Hepatic LD has a relatively high Km for lactate so only becomes significantly active when lactate concentration rises above a typical cellular level. 

The enzyme responsible for this topping-up ATP in active muscle is CK. CK is found in high concentration in muscle cells, both free within the sarcoplasm and also associated with membranes of mitochondria and the sarcoplasmic reticulum. Structurally, creatine kinase is a dimeric enzyme of B and/or M subunits, each of about 40 kDa. Three quaternary structure isoenzyme forms arise CK-MM, CK-BB and CK-MB. The predominant form in all muscles is CK-MM, but cardiac muscle also contains a significant amount of CK-MB and this isoenzyme can be used as a specific marker of myocardial damage (see Case Notes at the end of this chapter). 

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