Determination of creatine kinase activity and phosphocreatine in off-line and on-line modes with capillary electrophoresis
Introduction
Creatine kinase (CK) and its substrate phosphocreatine (PC) are analytes important to the human condition. The determination of CK activity has been used as a clinical tool for the investigation of skeletal muscle disease as well as for the diagnosis of myocardial infarction. In all types of muscular dystrophy, especially Duchenne type, CK activity is greatly elevated at 50 times the normal upper limit. Likewise, following myocardial infarction, CK activity in serum also begins to elevate within 4–6 h, with an average maximal elevation that is 7–12 times the upper normal limit [1]. In exercise science, interest stems from the fact that PC provides a reservoir of energy to sustain rapid muscle contraction. Muscle would soon exhaust its supply of adenosine triphosphate (ATP) if not for the thermodynamically favorable transfer of a phosphoryl group to adenosine diphosphate (ADP) which is catalyzed by CK [2]. Recently, the determination of PC has become a favorable marker in some conditioning programs for athletes [3]. Anaerobic ATP production in skeletal muscle is determined primarily by lactate accumulation and PC hydrolysis [4]. The assay for either CK activity or PC is often performed by spectrophotometry or fluorometry [5] following the production of NADH at 340 nm using a three enzyme coupled reaction scheme involving CK, hexokinase (HK), and glucose-6-phosphate dehydrogenase (GPDH).
Since the equilibrium constant (K) for the CK reaction has been reported as 1.4×108 but the K for GPDH reaction is 6.0×10−7, the production of NADH may not always accurately reflect the rate of ATP formation, particularly at high CK activity.
Past work by Huth and Danielson [6] demonstrated the use of high performance liquid chromatography (HPLC) for the off-line determination of CK enzyme activity at ml/min flow rates. In this work, samples were allowed to react for 13 min, quenched by heat, and then injected into the HPLC. Separation of the product ATP from ADP in about 16 min permitted the assay to be simplified to involve only the CK reaction. Besides determination of CK enzyme concentration in the range of 40–500 U/l, deproteinized serum samples of 12 and 304 U/l could be analyzed. No on-line HPLC work using just the CK reaction for the determination of CK activity has been reported to the best of our knowledge.
The applicability of using capillary electrophoresis (CE) to assay on-line for enzyme activities has been proven viable and reviews are available 7, 8. Both GPDH [9] and alcohol dehydrogenase [10] activities have been determined with CE by injecting the enzyme sample into a capillary filled with substrate and co-factor. Recently, enzyme catalyzed microreactions by CE have been conducted by sequentially injecting a plug of substrate and then an enzyme which will eventually meet and react [11]. In addition, previous work by us on the LDH enzyme system showed both activity and substrate assays using, respectively, injected sample or enzyme to initiate the reaction on-line in the capillary, could be made in less than 2 min using short capillaries [12]. For the substrate assays, the low consumption of injected enzyme stabilized with polyethylene glycol (PEG) is a major advantage of the CE approach. However, to the best of our knowledge, no study has been conducted examining any kinase enzyme and its substrate using CE both on-line and off-line in approach. In fact, any comparison of off-line and on-line enzyme assays involving CE is uncommon [8].
The objective of this research is to examine the CK enzyme system using CE for both the off-line and on-line determination of both the substrate, phosphocreatine (PC), as well as for CK activity. Only the reaction pathway involving CK will be used. First, results for the off-line determination of PC and CK activity in conjunction with CE will be presented. A comparison of two on-line CE methods for PC and CK activity will also be provided. The single injection on-line method is defined as when the enzyme or reagent sample to initiate the reaction is injected into the capillary filled with co-factor ADP and respective substrate sample or reagent. The on-line multiple injection protocol involves the successive injection of different mixtures of both the enzyme and substrate (one of which is the sample) into the capillary filled with buffer and co-factor. Advantages and disadvantages of both the off-line and on-line approaches will be discussed.
Section snippets
CE instrumentation and data collection
The CE instrument employed was a Thermal Separation (San Jose, CA) Spectrophoresis 500 equipped with a 20 cm×75 μm uncoated fused silica capillary (Polymicro, Phoenix, AZ) with an effective length of 15 cm to the detector window. Data acquisition was carried out with a Ranin Instrument (Woburn, MA) interface connecting the CE instrument to a MacIntosh SE computer with MacIntegrator I software. The CE detector time constant was at 0.5 s and data was acquired at a rate of 10 points per s. Detection
CK enzyme activity and stabilization
Previously, we have found that dehydrogenase enzymes when used as reagents for flow injection at μl/min flow rates can be stabilized with PEG [14] which can minimize subunit dissociation caused by solvent molecule interactions [15]. In the previous work, a 15% PEG solution was used but this was too viscous for CE. Addition of 1.5% PEG was considered to be optimal for CE to minimize the possibility of an irreversible blockage in the narrow capillary. An off-line CK enzyme assay was conducted
Conclusion
Comparing the on-line single and multiple injection techniques a dramatic improvement in throughput was established using the buffer method for both substrate and enzyme assays. A total of five substrate standards or enzyme samples could be assayed within 9 min. However, linearity particularly for CK activity was only fair and the multiple injection method may be more useful for a quick evaluation of CK enzyme levels or PC levels. The still automated on-line single injection method gives
Acknowledgements
Support by a NIH AREA grant is gratefully appreciated. We also thank Jennifer Cvrtnicek for doing some of the initial feasibility experiments.
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