Automated Fluorometric Creatine Kinase Assay. Measurement of 100-Fold Normal Activity without Serum Dilution

1974 ◽  
Vol 20 (5) ◽  
pp. 560-565 ◽  
Author(s):  
J B Armstrong ◽  
J A Lowden ◽  
A L Sherwin
Keyword(s):  
Creatine Kinase ◽  
Normal Activity ◽  
Normal Serum ◽  
Kinase Assay ◽  
Alkaline Ph ◽  
Protein Fluorescence ◽  
Severe Renal Failure ◽  
Additional Advantage ◽  
Serum Dilution ◽  
Sample Dilution

Abstract Creatine kinase (EC 2.7.3.2) activity can be measured fluorometrically by coupling the reaction product, creatine, to ninhydrin at alkaline pH. We have automated this procedure to develop an accurate and precise method, which requires 0.1 ml of sample and can be performed at 60 tests per hour. By using an abbreviated incubation time and a dialyzer in the assay, we have extended the analytical range to 100 times normal serum activity without sample dilution. The dialyzer also eliminates the nonspecific protein fluorescence of the sample. An additional advantage of the method is the reduced cost of reagents in comparison to similar procedures. Serum blanks should be analyzed whenever serum creatine is abnormally high, especially in cases of severe renal failure.

Infantile polymyositis with normal serum creatine kinase level

1997 ◽  
Vol 19 (1) ◽  
pp. 63-65 ◽  
Author(s):  
Sarenur Tütüncüogˇlu ◽  
Hasan Tekgül ◽  
Eren Demirtas¸ ◽  
Seval Uysal
Keyword(s):  
Creatine Kinase ◽  
Creatine Kinase Level ◽  
Normal Serum ◽  
Serum Creatine Kinase ◽  
Serum Creatine Kinase Level

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

1976 ◽  
Vol 22 (7) ◽  
pp. 1078-1083 ◽  
Author(s):  
T G Rosano ◽  
K J Clayson ◽  
P E Strandjord
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Creatine Kinase Activity ◽  
Serum Creatine Kinase ◽  
Assay System ◽  
Kinase Assay ◽  
Rabbit Muscle ◽  
Atp Production

Abstract Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.

Creatine kinase and lactate dehydrogenase: stability of isoenzymes and their activity in stored human plasma and prostatic tissue extracts and effect of sample dilution.

1983 ◽  
Vol 29 (5) ◽  
pp. 832-835 ◽  
Author(s):  
S A Shain ◽  
R W Boesel ◽  
R W Klipper ◽  
C M Lancaster
Keyword(s):  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Human Plasma ◽  
Enzyme Activities ◽  
Prostatic Tissue ◽  
Tissue Extracts ◽  
Sample Dilution ◽  
Analytical Protocols

Abstract We examined the effect of storing human plasma or extracts of prostate at -90 degrees C on the activity of creatine kinase and lactate dehydrogenase and isoenzyme distribution. Enzyme activities were unaltered during storage for as long as six weeks. If these preparations were thawed only once at 2 to 4 degrees C, they could be stored for as long as 165 days at -90 degrees C with no change in isoenzyme distribution. Inexplicably, apparent isoenzyme distribution of prostatic lactate dehydrogenase was sensitive to sample dilution, whereas the isoenzyme distribution of lactate dehydrogenase in plasma was not. Our observations emphasize the importance of validating details of analytical protocols that are to be used for quantification of new types of specimens.

Activation of human creatine kinase isoenzymes by pH and various sulfhydryl and chelating agents.

1981 ◽  
Vol 27 (3) ◽  
pp. 402-404 ◽  
Author(s):  
D A Nealon ◽  
S M Pettit ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Ethylene Glycol ◽  
Chelating Agents ◽  
Enzyme Stability ◽  
Kinase Assay ◽  
Lag Phase ◽  
Creatine Kinase Isoenzymes

Abstract We report the effect of serum pH and of the presence or absence of mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate on the activation of the human creatine kinase isoenzymes. At the serum pH giving maximal enzyme stability and minimal assay lag phase (Nealon et al., Clin. Chem. 26: 1165-1169, 1980) thiol activation of CK-1 and CK-3 is nearly maximal with monothioglycerol in an optimized creatine kinase assay (Szasz et al., Clin. Chem. 22: 650-656, 1976). However, CK-2 is maximally activated at pH 8.5, a pH at which this isoenzyme is least stable on storage and its assay lag phase is prolonged. These findings suggest irreconcilable problems in the storage, activation, and assay of CK-2.

Single Stable Reagent for Creatine Kinase Assay

1971 ◽  
Vol 17 (6) ◽  
pp. 548-550 ◽  
Author(s):  
Charles Bishop ◽  
T M Chu ◽  
Z K Shihabi
Keyword(s):  
Creatine Kinase ◽  
Maximum Velocity ◽  
Creatine Phosphate ◽  
Spectrophotometric Assay ◽  
Kinase Assay ◽  
Reagent Cost ◽  
One Step ◽  
Stable Reagent

Abstract A single stable reagent for spectrophotometric assay of creatine kinase in one step is described. Reagent cost has been decreased by running the reaction at Km conditions for creatine phosphate and relating the activity to maximum velocity.

Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes.

1980 ◽  
Vol 26 (8) ◽  
pp. 1165-1169 ◽  
Author(s):  
D A Nealon ◽  
S M Pettit ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Ethylene Glycol ◽  
Storage Stability ◽  
Kinase Assay ◽  
Lag Phase ◽  
Creatine Kinase Isoenzyme ◽  
Sh Groups ◽  
C Storage ◽  
Creatine Kinase Isoenzymes

Abstract We report the effect of serum pH on the storage stability of the human creatine kinase isoenzymes and on the creatine kinase assay lag phase (Szasz et al., Clin. Chem. 22: 650, 1976). We also investigated the effect of including mercaptoethanol, N-acetyl-L-cysteine, monothioglycerol, ethylenediaminetetraacetate, or ethylene glycol bis(betaaminoethyl ether)-N,N,N',N'-tetraacetate at 20, 4, and --20 degrees C. Storage stability of the isoenzymes is profoundly affected by pH. For patients' samples and semi-purified human creatine kinase isoenzymes added to heat-inactivated sera, increasing diluent pH above 7.0 decreases creatine kinase stability. The thiol agents or chelators generally give little or no protection above pH 7.5; at pH 8.5 they contribute significantly to isoenzyme instability. Storage at 4 degrees C provides greater stability than storage at 20 degrees C, particularly in the case of creatine kinase isoenzyme BB. The lag phase was minimum at a serum pH of 6.5, in the presence of 10 mmol of monothioglycerol per liter. Increasing serum pH to 8.5 prolongs the reaction lag phase by about 1 min over the minimum. We recommend that, before they are stored at 4 degrees C, the pH of patients' samples be adjusted to 6.5 and oxidation of SH-groups be minimized by adding monothioglycerol to the sample.

Spurious brain creatine kinase in serum from patients with renal disease.

1978 ◽  
Vol 24 (9) ◽  
pp. 1636-1638 ◽  
Author(s):  
R B Coolen ◽  
R Herbstman ◽  
P Hermann
Keyword(s):  
Renal Failure ◽  
Creatine Kinase ◽  
Renal Disease ◽  
Normal Serum ◽  
Disease States ◽  
Low Concentrations ◽  
Brain Creatine Kinase ◽  
End Stage ◽  
Increased Activity

Abstract Creatine kinase isoenzyme I(BB) is generally not detectable in normal serum, and its occurrence in serum has been documented in only a few disease states. In particular, increased activity of this isoenzyme has been reported in association with chronic renal failure, hemodialysis, and renal transplantation. The present study demonstrates that the apparent creatine kinase observed in the serum of such renal patients is an artifact, observed as a result of measuring creatine kinase isoenzymes by fluorescence. Our observations resemble those of McKenzie et al. [Clin. Chim. Acta 70, 333(1976)] concerning an artifact in the fluorometric determination of lactate dehydrogenase isoenzymes in the sera of patients with end-stage renal failure. The artifact binds to albumin, is not a protein, and occurs in some normal sera at very low concentrations. This artifact can be mistakenly identified as isoenzyme I in renal-disease patients if CK isoenzymes are determined fluorometrically.

Creatine kinase in serum: 2. Interference of adenylate kinase with the assay.

1976 ◽  
Vol 22 (11) ◽  
pp. 1806-1811 ◽  
Author(s):  
G Szasz ◽  
W Gerhardt ◽  
W Gruber ◽  
E Bernt
Keyword(s):  
Creatine Kinase ◽  
Adenylate Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Hepatic Damage ◽  
Complete Inhibition ◽  
Kinase Assay ◽  
Competitive Inhibitors ◽  
Diadenosine Pentaphosphate ◽  
Adenylate Kinases

Abstract Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%.

Transformation of creatine kinase-BB isoenzyme in vitro: effect of carboxylic acids, thiols, pH, cations, and chelators.

1982 ◽  
Vol 28 (12) ◽  
pp. 2414-2417 ◽  
Author(s):  
J A Lott ◽  
J W Heinz
Keyword(s):  
Creatine Kinase ◽  
Rat Brain ◽  
Carboxylic Acids ◽  
Alkaline Ph ◽  
Creatine Kinase Isoenzyme ◽  
Creatine Kinase Bb ◽  
Vitro Effect ◽  
Dialyzed Serum

Abstract Creatine kinase isoenzyme BB from rat brain was incubated with serum, dialyzed serum, or plasma, with added carboxylic acids, thiols, buffers, or cations. It was found to be very unstable at 37 degrees C under all these conditions, being transformed to CK-BB', a form that migrates like CK-MB on agarose electrophoretic plates. This transformation is enhanced at alkaline pH and, especially, by Zn2+. CK-BB' is quite stable, and probably results from binding of cations to CK-BB, because the transformation is prevented by EDTA and citrate.

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