Demonstration of serum creatine kinase isoenzymes by fluorescence technique

Somer, Hannu; Konttinen, Aarne

doi:10.1016/0009-8981(72)90260-4

Cited by 71 publications

(20 citation statements)

References 10 publications

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“…It can be seen that MB activity always is under 30% of total CPK activity, in accordance with data obtained with the electrophoresis technique (7)(8)(9). Human myocardial tissue contains about 30% MB isoenzyme (6) and this study thus shows that the necrotic tissue probably releases both MM and MB CPK at the same rate and to the same extent.…”

Section: Discussionsupporting

confidence: 76%

“…(6), the appearance of MB enzyme in the serum is useful for diagnosing myocardial damage. Normal human serum contains only the MM isoenzyme while the MB enzyme appears in serum from patients after an acute myocardial infarction (7)(8)(9). In the present study we have utilized known differences in the kinetic properties of the isoenzymes to detect the MB isoenzyme in serum from such patients with a new procedure not requiring electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic Properties of the Isoenzymes of Human Creatine Phosphokinase

Witteveen

Sobel

DeLuca

1974

Proc. Natl. Acad. Sci. U.S.A.

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Studies of the three human creatine phosphokinase (EC 2.7.3.2) isoenzymes, MM, MB, and BB,show that important differences exist in substrate dependency of the reaction rates. A method was developed to study these properties in which the ATP formed in the reverse reaction was measured by means of firefly luciferase. With substrate conditions at which the isoenzymes showed substantial differences in activity the method could be used for the detection of changes in the-isoenzyme pattern of the' serum of patients with an acute myocardial infarction. The MB isoenzyme appearing in this condition could be detected quantitatively. (6), the appearance of MB enzyme in the serum is useful for diagnosing myocardial damage. Normal human serum contains only the MM isoenzyme while the MB enzyme appears in serum from patients after an acute myocardial infarction (7)(8)(9). In the present study we have utilized known differences in the kinetic properties of the isoenzymes to detect the MB isoenzyme in serum from such patients with a new procedure not requiring electrophoresis.The method conventionally used for the detection of CPK activity in sera is that of Rosalki (10) and is a coupled enzyme assay. However, this assay cannot be used at very low concentrations of substrates due to a lack of sensitivity. Since we wished to assay CPK at very low substrate concentrations where the difference in activity of the isoenzymes is most apparent, we developed a kinetic method in which the ATP produced is measured by the firefly light-emitting reaction. Thus the reaction CP + ADP = creatine + ATP is followed by coupling the ATP formed with the firefly luciferase reaction in which E ATP + luciferin + 02 --oxyluciferin + AMP + PPi + CO2 + h., where E is luciferase.Under selected conditions the light emission at any moment is proportional to the amount of ATP present at that instant.When CPK is present in the reaction medium, and its substrates, ADP and CP, are added, the rate of increase of emission of light is proportional to the rate of formation of ATP during a measured interval. By using the Rosalki method for CPK activity at high substrate concentrations and the luciferase-coupled assay at low substrate concentrations, it is possible to determine the amount of the three isoenzymes present in a sample. We have used this technique to study the changes in serum CPK isoenzymes in patients with acute myocardial infarction. The data indicate that this is a rapid and sensitive method for detecting the appearance of MB isoenzyme in serum. MATERIALS AND METHODSReagents. Total CPK activity was assayed according to Rosalki (10) using Calbiochem Statpacks. CP and ADP were purchased from Calbiochem. ADP was further purified by chromatography on a DEAE-Sephadex A-25 column to remove ATP contaminants. Luciferase was purified as described by Green and McElroy (11)

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Section: Discussionsupporting

confidence: 76%

Section: Introductionmentioning

confidence: 99%

Kinetic Properties of the Isoenzymes of Human Creatine Phosphokinase

Witteveen

Sobel

DeLuca

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…The unstained creatine kinase bands corresponding to creatine kinase-MM and creatine kinase-MB were cut out and layered carefully onto the surface of the thin layer isoelectric focusing gel. The corresponding serum samples (3)(4) were also loaded onto the thin layer gel. All samples were focused and stained äs described in "methods".…”

Section: Between-ruii-precisionmentioning

confidence: 99%

The Isoelectric Focusing of Creatine Kinase Variants: II. The Heterogeneity of Creatine Kinase in Human Serum with Normal and Elevated Catalytic Concentrations

SiragEldin¹,

Gercken²,

Harm³

1986

Clinical Chemistry and Laboratory Medicine

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“…After an infarct it first appears in the plasma within four hours (Roberts et al, 1977) and reaches a peak at between J6 and 24 hours (Wagner et al, 1973); the levels fal1 to normal after 48 hours (Roe et al, 1972). Plasma activity remains low after cardioversion (Konttinen and Somer, 1973), coronary angiography (Wagner et al, 1973), pulmonary embolus (Somer and Konttinen, 1972), angina pectoris (Wolf and Kearns, 1974), arrythmias or pericarditis (Fiolet et al, 1977), intramuscular injection (Rao et al, 1975;Roberts et al, 1975;Ahumada et al, 1976), or exercise (Kaman et al, 1977). CK-MB activity is detectable in normal plasma, but only at low activity, and the conditions other than myocardial infarction (MI), in which it is significantly increased, such as Duchenne muscular dystrophy (Konttinen and Somer, 1973), crush injury (Wilhelm and Todd, 1977), and acromegalic myopathy (Wolf and Griffith, 1978), are not usually considered in the differential diagnosis of Ml.…”

mentioning

confidence: 99%

A Biochemical and Clinical Comparison of two Commercially Available Creatine Kinase Iso-Enzyme MB Assay Kits Suitable for use in the Routine Medical Laboratory

Surtees¹,

Bastable²,

Gietzen³

et al. 1979

Ann Clin Biochem

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SUMMARYTwo methods for the measurement of plasma creatine kinase MB (CK-MB) activity were compared for analytical performance, cost, practicality, and diagnostic correlation with clinical and electrocardiographic findings in patients admitted to the coronary care unit of a district general hospital. The methods were column chromatography and immunoinhibition. Both methods were found acceptable, and the method to be adopted would depend on the staff arrangements and resources available in the laboratory.

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Demonstration of serum creatine kinase isoenzymes by fluorescence technique

Cited by 71 publications

References 10 publications

Kinetic Properties of the Isoenzymes of Human Creatine Phosphokinase

Kinetic Properties of the Isoenzymes of Human Creatine Phosphokinase

The Isoelectric Focusing of Creatine Kinase Variants: II. The Heterogeneity of Creatine Kinase in Human Serum with Normal and Elevated Catalytic Concentrations

A Biochemical and Clinical Comparison of two Commercially Available Creatine Kinase Iso-Enzyme MB Assay Kits Suitable for use in the Routine Medical Laboratory

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