Isoenzymes measurement

A number of immunoassays for human enzymes and isoenzymes measuring protein mass instead of catalytic activity... 

Galen RS, Reiffel JA, Gambino SR. Diagnosis of acute myocardial infarction relative efficiency of serum enzyme and isoenzyme measurements. JAMA 1975 232 145-7. 

Ion-exchange HPLC separation of enzymes has been a particularly active area of research, mainly because of the clinical usefulness of isoenzyme measurements. Most separations employ a conventional colorimetric or fluorometric assay on the eluent (post-column) to detect resolved isoenzymes (F9). 

Fluorescence ascribed to CK-BB intensity correlated with the duration of renal failure CK isoenzyme measurement by fiuorogenic substrate... 

Fluorescent material different from CK-BB, with electrophoretic mobility similar but somewhat slower than that of CK-BB Electrophoresis of CK isoenzymes, measuring fluorescence of NADPH... 

CK Originates from cardiac muscle, skeletal smooth muscle and brain. Isoenzyme measurements used, paliculaily CK-MB in myocardial inlaiclion... 

Alkaline phosphatase Isoenzyme measurements may help to distinguish between bone, liver. Intestinal and placental fractions. Rais during fracture healing, late pregnancy and in children during rapid growth ... 

The isoenzymes associated with heart muscle can convert a-oxobutyrate (which has a similar structure to pyruvate) to a-hydroxybutyrate more effectively than the other LDH isoenzymes. Measurement of serum hydroxybutyrate dehydrogenase activity therefore gives an estimate of heart LDH (see separate entry on hydroxybutyrate dehydrogenase). 

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. 

Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

HBD is a biochemical rather than electrophoretic assessment of the LD isoenzyme which is associated with heart. All five isoenzymes of LD exhibit some activity toward cx-hydroxy-butyrate as substrate, but heart LD shows the greatest activity. Serum HBD measurement is not as valuable as the electrophoretic determination of heart LD isoenzyme. High HBD activity has also been found in diseases of the liver. Rises associated with the hepatic effects of congestive heart failure can be disconcerting in the differential diagnosis of myocardial infarction. Wilkinson has used the serum HBD/LD ratio for the differentiation of myocardial disease from other disorders in which HBD activity is elevated, whereas Rosalki has not found the ratio to be helpful (39). 

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

Time-resolved approaches for multi-analyte immunoassays have been described recently. Simultaneous determination of LH, follicle stimulating hormone (FSH), hCG, and prolactin (PRL) in a multisite manual strip format has been reported. 88 Four microtiter wells are attached to a plastic strip, two-by-two and back-to-back, such that the wells can be read on a microtiter plate reader. In a quadruple-label format, the simultaneous quantitative determination of four analytes in dried blood spots can be done using europium, samarium, dysprosium, and terbium. 89 In this approach, thyroid-stimulating hormone, 17-a-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM (CK-MM) isoenzyme are determined from dried blood samples spotted on filter paper in a microtiter well coated with a mixture of antibodies. Dissociative fluorescence enhancement of the four ions using cofluorescence-based enhancement solutions enables the time-resolved fluorescence of each ion to be measured through four narrow-band interference filters. 

A spectrophotometric assay procedure was used to measure the lipoxygenase isoenzyme activities of fresh and stored soya containing bread improver pastes and powders (77). 

Administration of hydroquinone (0.5% in the diet) for 20 weeks did not induce hyperplasia or papillomatous lesions in the forestomach in Syrian golden hamsters (Hirose etal., 1986). In male Fischer 344 rats, oral administration of hydroquinone for eight weeks (0.8% in the diet) did not induce hyperplasia or DNA synthesis, as measured by BrdU-labelling index in the forestomach epithelium. No cell proliferation, increased DNA synthesis or increase in pepsinogen-isoenzyme-1-altered neoplastic foci was observed in the pyloric mucosa (Shibata et al., 1990). 

Very recently the isolation of still active carbonic anhydrase from the biocrystalline layer of hens egg shells has been reported. Crude egg shell extracts exhibited no enzyme activity. however, after removal of Ca2+ and simple gel filtration of the extracts, carbonic anhydrase activity could be measured. Further purification steps led to the isolation of two isoenzymes. The possibility of inhibition of this enzyme activity by shell components cannot be excluded although recombination of shell components after separation did not inhibit that enzyme. The isolated enzymes has an apparent molecular size of 28,000 daltons. Carbonic anhydrase from egg shells catalyzes the reversible hydration of CO2 + HOH H+ + HCO3-. This probably is the primary action of the enzyme of the shell. Moreover, egg shell carbonic anhydrase catalyzes the hydration of acetaldehyde and pyridine aldehyde. Furthermore, the same enzymes have esterase activity (hydrolysis of p-nitrophenyl acetate). Whether the latter activities play a role in the egg shell cannot be judget at the present time. 

Fig. 4. Changes in A, GS mRNAs and B, GS isoenzymes, during nodule development. The abundances of the mRNAs were measured by an RNase protection technique and the isoenzymes following separation by IEX-HPLC (adapted from Cullimore et al., 1990). GSs=GS synthetase activity. The isoenzymes are as defined in Fig. 3.

Several species and populations from other Anopheles complexes have been discriminated based on CHC patterns. Examples include all five species of the An. quad-rimaculus complex (Carlson et al., 1997), some species of the An. maculipennis complex (Phillips et al., 1990a), malaria-vector and non-vector forms of the An. maculates complex (Kittayapong et al., 1990, 1993), and An. Stephensi strains susceptible or resistant to DDT and malathion (Anyanwu et al., 1993, 1997). CHCs have been used in combination with isoenzyme analysis to successfully differentiate populations of An. darlingi (Rosa-Freitas et al., 1992). All these findings demonstrate that hydrocarbon analysis is a powerful tool for distinguishing mosquito species and populations. This is particularly important for disease vectors, since it can facilitate interpretation of epidemiological data and assist implementation of control measures. 

Measured at 401 nm for acidic isoenzymes and 403 nm for neutral and basic isoenzymes... 

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. 

CK is found primarily in heart and skeletal muscle, as well as the brain. Measurement of serum CK levels is a good diagnostic for injury to these tissues. Levels of CK rise within 6 hours of injury and peak by around 18 hours, returning to normal within 2-3 days. Like LDH, there are tissue-specific isoenzymes of CK ... 

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